Pharmaceutical composition for reducing local fat and uses thereof

ABSTRACT

The present invention provides a pharmaceutical composition for reducing localized fat, comprising drug-containing micelles made of surfactants, and curcumin encapsulated in said drug-containing micelles. This pharmaceutical composition for reducing localized fat can reduce the fat at the administration site, and has the advantages of high stability, high bioavailability for fat tissue, few side effects, and sustained release.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition forreducing localized fat, specifically, relates to a pharmaceuticalcomposition comprising drug-containing micelles and curcuminencapsulated in the micelles, and the pharmaceutical composition is forlocalized fat reduction.

BACKGROUND OF THE INVENTION

In recent years, more and more people have changed their views of beautyand raised their standards of individual health and body shape. As aresult, people are not only concerned about weight loss, but focus moreon reducing localized fat or contouring body shape in order to behealthier and achieve better body shape. Furthermore, common weight lossregimens, whether it is through diet or exercise, cannot reduce fat at aspecific location. Currently, if wanting to reduce localized fat atspecific locations (such as the waist, abdomen, legs, arms, chin, andface, etc.), the only available technology is liposuction.

At present, liposuction is the most prevalent technology to reducelocalized fat. However, the procedure of liposuction causes severedamages to the nerves, blood vessels, and other tissues. Liposuctionalso comes with risks of infections, severe bleeding, prolongedanesthesia, and unpredictable life-threatening conditions such as fatembolism and allergic reactions to anesthesia. In addition, it is commonto experience significant bruising and swelling, severe pain, andpost-operational recovery can take as long as 3 to 6 months, and theliposuction site may become uneven. Therefore, statistical analysesrevealed that even though many people have considered liposuction toreduce the accumulation of localized subcutaneous fat or improve bodycurves, less than 40 percent of them actually went through liposuction.It shows that the customers who want to improve body curves or reducelocalized fat are deterred from the problems of side effects ofliposuction, pain after liposuction, or risks of liposuction.

Although some non-surgical localized fat-reducing pharmaceuticalcompositions or equipments can partially lower the side effects, theyare usually not effective and come with other side effects, such asnecrosis of surrounding normal cells, inflammation of surroundingtissues, and sharp pain. Additionally, their administration sites arelimited. Therefore, the market is eagerly demanding for an effectivelocalized fat-reducing pharmaceutical composition that has less sideeffects, a better safety profile, and a shorter recovery period.

In case of the customers and doctors have significant high demand, thepressing concern is to develop a localized fat-reducing pharmaceuticalcomposition that breaks through the limits of current technologies.

SUMMARY OF THE INVENTION

In view of the deficiency of prior arts, the present invention providesa pharmaceutical composition for reducing localized fat, comprisingdrug-containing micelles made of surfactants, and curcumin encapsulatedin said drug-containing micelles. The pharmaceutical composition forreducing localized fat can reduce the fat at the administration site,and has the advantages of high stability, high fat tissuebioavailability, few side effects, and sustained release.

The present invention can promote apoptosis of the local adipocytes atthe administration sites, thereby achieving the goal of reducinglocalized fat. The present invention drastically improves the adversereactions and side effects of the prior arts such as necrosis of thesurrounding cells and inflammation reactions. The present invention issuitable for direct injection, subcutaneous implantation, intravenousinjection, implanted infusion, cream, patch, and other skin absorptionsystemic delivery methods to administer at the sites requiring fatreduction without the need or assistance of any surgery or equipment.Preferably, it is administered locally at the subcutaneous fat layer viasubcutaneous injection. Preferably, the injection formulation of thepresent pharmaceutical composition includes but is not limited to powderfor injection, or powder for solution for injection. The localized fatmentioned herein includes but is not limited to the fat at the waist,the legs, the arms, the chin, and the face.

In the present invention, the term “turmeric extract” refers to thecurcumin ingredient mixture extracted by any solvent and any extractionmethod, commercially available turmeric extract, any mixture containingat least 75% (wt %) of curcumin, any mixture containing at least 75% (wt%) of curcuminoid, or commercially available curcumin.

Wherein, curcuminoid is a collective term for curcumin,demethoxycurcumin, and bisdemethoxycurcumin.

In the present invention, the term “resveratrol” refers to theresveratrol extracted from natural plants or commercially availableresveratrol. Preferably, the purity of resveratrol is 90% to 100% (wt%).

In the present invention, the term “green tea extract” refers to thegreen tea ingredient mixture extracted by any solvent and any extractionmethod, commercially available green tea extract, any mixture containingat least 45% of epigallocatechin gallate (EGCG), or commerciallyavailable epigallocatechin gallate (EGCG).

In the present invention, the term “micelle” refers to a microstructureformed by surfactants, wherein each of the surfactants has a hydrophilicend and a hydrophobic (lipophilic) end, and the surfactants are arrangedin a way that the hydrophilic ends face outward and the hydrophobic(lipophilic) ends face inward to form the microstructure. Preferably,the microstructure is a spherical structure, a spheroidal structure, orother microstructural structures.

In the present invention, the term “drug-containing micelles” refer tothe micelles containing curcuminoid. Preferably, drug-containingmicelles refer to the micelles containing curcumin; that is,drug-containing micelles refer to the micelles encapsulating orcontaining curcuminoid. Preferably, drug-containing micelles refer tothe micelles encapsulating or containing curcumin.

In the present invention, the term “second lipophilic drug-containingmicelles” refer to the micelles containing any lipophilic drug exceptcurcuminoid. That is, the second lipophilic drug-containing micellesrefer to the micelles encapsulating or containing the second lipophilicdrug.

Wherein, the term “other lipophilic drug (or the second lipophilicdrug)” refers to at least one of quercetin, synephrine, puerarin,resveratrol, and any lipophilic drug except curcuminoid, or combinationthereof. Or, the other lipophilic drug refers to any lipophilic drugexcept curcumin.

In the present invention, the term “hydrophilic drug” refers to at leastone of green tea extract, epigallocatechin gallate, epicatechin,epicatechin gallate, epigallocatechin, gallocatechin gallate,gallocatechin, catechin gallate, catechin, epigallocatechin gallate(EGCG), caffeine, carnitine, L-carnitine, synephrine, chlorogenic acid,and other hydrophilic drugs, or combination thereof.

In the present invention, the term “state without precipitation”, asused herein, refers to a state wherein no precipitation can be observedwith the naked eye, that is, without the need by the assistance ofartificial instruments.

In the present invention, the term “localized subcutaneous fat” refersto the subcutaneous fat at the site administered with the pharmaceuticalcomposition, subcutaneous injection formulation, or subcutaneous fatlayer injection formulation of the present invention.

In the present invention, the term “pharmaceutically acceptablesolution” is at least one of water for injection, aqueous solution forinjection, and normal saline, or combination thereof.

In the present invention, the term “local anesthetic” is at least one ofamides, para-aminobenzoic acid esters, amino ethers, and other localanesthetic, or combination thereof. Preferably, the amides are at leastone of dibucaine, lidocaine, mepivacaine HCl, bupivacine HCl, pyrrocaineHCl, prilocaine HCl, digammacaine, and oxethazaine, or combinationthereof. Preferably, the para-aminobenzoic acid esters are at least oneof butacaine, dimethocaine, and tutocaine, or combination thereof.Preferably, the amino ethers are at least one of quinisocaine andpramocaine, or combination thereof.

In the present invention, the term “antioxidant” is at least one ofbeta-carotene, lutein, lycopene, bilirubin, vitamin A, vitamin C(ascorbic acid), vitamin E, uric acid, nitric oxide, nitoxide, pyruvate,catalase, superoxide dismutase, glutathione peroxidases, N-acetylcysteine, naringenin, and other antioxidant, or combination thereof.

In the present invention, the pharmaceutical composition maintains at astate without precipitation for at least 24 hours when it is subjectedto accelerated stability test at 25° C.±2° C. relative humidity 60%±5%,and in the absence of direct light.

Or, the pharmaceutical composition maintains at a state withoutprecipitation for at least 6 months when it is subjected to acceleratedstability test at 25° C.±2° C., relative humidity 60%±5%, and in theabsence of direct light.

The present invention provides a pharmaceutical composition to beadministered at a local site of a subject, comprising:

-   -   drug-containing micelles; and    -   curcuminoid encapsulated in said drug-containing micelles;        wherein, said drug-containing micelles are a microstructure        formed by a pharmaceutically acceptable polyoxyethylene castor        oil derivative, and the hydrophilic-lipophilic balance value        (HLB value) of the polyoxyethylene castor oil derivative is        greater than 10.

Preferably, the pharmaceutical composition further comprises apharmaceutically acceptable aqueous solution, and said drug-containingmicelles are evenly distributed in said pharmaceutically acceptableaqueous solution.

Preferably, the polyoxyethylene castor oil derivative is at least one ofKOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP), KOLLIPHOR® RH 40 (polyoxyl 40 hydrogenated castor oil), and otherpolyoxyethylene castor oil derivatives, or combination thereof.

Preferably, the weight ratio of the curcuminoid to the polyoxyethylenecastor oil derivative is 1:8-1:500.

Preferably, the weight ratio of the curcuminoid to the polyoxyethylenecastor oil derivative is 1:20-1:150.

Preferably, the concentration of curcuminoid in the pharmaceuticalcomposition is 0.3-120 mg/g.

Preferably, the concentration of curcuminoid in the pharmaceuticalcomposition is 2-91 mg/g.

Preferably, the diameter of the drug-containing micelles is 3-50 nm.

Preferably, the diameter of the drug-containing micelles is 5-20 nm.

Preferably, the curcuminoid is curcumin.

Preferably, the pharmaceutical composition further comprises secondlipophilic drug-containing micelles, and the second lipophilicdrug-containing micelles are evenly distributed in the pharmaceuticallyacceptable aqueous solution; the second lipophilic-drug containingmicelle is a second microstructure formed by a second polyoxyethylenecastor oil derivative, and a second lipophilic drug is encapsulated insaid second drug-containing micelles.

Preferably, the hydrophilic-lipophilic balance value (HLB value) of thesecond polyoxyethylene castor oil derivative is greater than 10.

Preferably, the second polyoxyethylene castor oil derivative is at leastone of KOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known asCREMOPHOR® ELP), KOLLIPHOR® RH 40 (polyoxyl 40 hydrogenated castor oil),and other polyoxyethylene castor oil derivatives, or combinationthereof.

Preferably, the second lipophilic drug is at least one of quercetin,synephrine, puerarin, resveratrol, and any other lipophilic drug exceptcurcuminoid, or combination thereof.

Preferably, the weight ratio of the curcuminoid to the second lipophilicdrug is 30:1-1:10.

Preferably, the weight ratio of the curcuminoid to the second lipophilicdrug 20:1-1:8.

Preferably, the pharmaceutically acceptable aqueous solution furthercomprises a hydrophilic drug.

Preferably, the hydrophilic drug is at least one of green tea extract,epigallocatechin gallate, epicatechin, epicatechin gallate,epigallocatechin, gallocatechin gallate, gallocatechin, catechingallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine,L-carnitine, synephrine, chlorogenic acid, and other hydrophilic drugs,or combination thereof.

Preferably, the weight ratio of the curcuminoid to the hydrophilic drugis 30:1 to 1:10.

Preferably, the concentration of epigallocatechin gallate (EGCG) in thepharmaceutical composition is 0.1-15 mg/mL.

Preferably, the pharmaceutical composition further comprises a cosolventto increase the solubility of drugs.

Preferably, the cosolvent is at least one of polyethylene glycol,propylene lycol, ethanol, and other cosolvents, or combination thereof.

Preferably, the polyethylene glycol is at least one of PEG 200, PEG 400,PEG 600, and other polyethylene glyclols, or combination thereof.

Preferably, the pharmaceutical composition further comprises asuspending agent to reduce the sedimentation rate of drugs or micelles.

Preferably, the suspending agent is at least one of sodium alginate,glycerol, carboxymethylcellulose sodium, mannitol, and other suspendingagents, or combination thereof.

Preferably, the pharmaceutical composition further comprises an oilphase excipient to increase the stability of the pharmaceuticalcomposition and the solubility of drugs.

Preferably, the oil phase excipient is at least one of unsaturated fattyacids, glycerol, triglycerides, and other oil phase excipients, orcombination thereof.

Preferably, the unsaturated fatty acids are at least one of oleic acid,castor oil, sesame oil, cottonseed oil, soybean oil, safflower oil, cornoil, and other unsaturated fatty acids, or combination thereof.

Preferably, the triglycerides are at least one of medium chaintriglycerides, and other triglycerides, or combination thereof.

Preferably, the pharmaceutical acceptable aqueous solution comprises alocal anesthetic.

Preferably, the pharmaceutically acceptable aqueous solution comprisesan antioxidant.

The present invention further provides a use of the pharmaceuticalcomposition in preparing a subcutaneous injection formulation, asubcutaneous fat layer injection formulation, a subcutaneous implanteddevice, a subcutaneous implant, an intravenous injection formulation, asolution for implanted infusion, a cream, a patch, or otherskin-absorption delivery systems.

Preferably, the pharmaceutical composition further comprises secondlipophilic drug-containing micelles, and the second lipophilicdrug-containing micelles are evenly distributed in the pharmaceuticallyacceptable aqueous solution; wherein, the second lipophilicdrug-containing micelle is a second microstructure formed by a secondnon-ionic surfactant, and a second lipophilic drug is encapsulated insaid second lipophilic drug-containing micelles.

Preferably, the second non-ionic surfactant is at least one ofpolysorbate 80 (TWEEN® 80), polyoxyl 15 hydroxystearate (KOLLIPHOR® HS15), polyoxyethylene castor oil derivatives, and other non-ionicsurfactants, or combination thereof.

Preferably, the polyoxyethylene castor oil derivative is at least one ofKOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP). KOLLIPHOR® RH 40 (polyoxyl 40 hydrogenated castor oil, formerlyknown as CREMOPHOR® RH 40), and other polyoxyethylene castor oilderivatives, or combination thereof.

Preferably, the pharmaceutically acceptable aqueous solution furthercomprises a hydrophilic drug.

The present invention further provides a use of the pharmaceuticalcomposition in preparing a drug or a subcutaneous injection formulationfor reducing localized subcutaneous fat; the pharmaceutical compositioncomprises:

-   -   drug-containing micelles; and    -   curcuminoid encapsulated in said drug-containing micelles;        wherein, the drug-containing micelle is a microstructure formed        by a pharmaceutically acceptable non-ionic surfactant, and the        hydrophilic-lipophilic balance value (HLB value) of the        non-ionic surfactant is greater than 10.

Preferably, the pharmaceutical composition further comprises apharmaceutically acceptable aqueous solution, and said drug-containingmicelles are evenly distributed in said pharmaceutically acceptableaqueous solution.

Preferably, the non-ionic surfactant is at least one of polysorbate 80(TWEEN® 80), polyoxyl 15 hydroxystearate (KOLLIPHOR® HS 15),polyoxyethylene castor oil derivatives, and other non-ionic surfactants,or combination thereof.

Preferably, the polyoxyethylene castor oil derivative is at least one ofKOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP), KOLLIPHOR® RH40 (polyoxyl 40 hydrogenated castor oil, formerlyknown as CREMOPHOR® RH 40), and other polyoxyethylene castor oilderivatives, or combination thereof.

Preferably, the weight ratio of the curcuminoid to the non-ionicsurfactant is 1:8 to 1:500.

Preferably, the concentration of curcuminoid in the pharmaceuticalcomposition is 0.3-120 mg/g.

Preferably, the diameter of the drug-containing micelles is 3-50 nm.

Preferably, the pharmaceutical composition further comprises secondlipophilic drug-containing micelles, and the second lipophilicdrug-containing micelles are evenly distributed in the pharmaceuticallyacceptable aqueous solution; wherein, the second lipophilicdrug-containing micelle is a second microstructure formed by a secondnon-ionic surfactant, and a second lipophilic drug is encapsulated insaid second lipophilic drug-containing micelles.

Preferably, the hydrophilic-lipophilic balance value (HLB value) of thesecond non-ionic surfactant is greater than 10.

Preferably, the second non-ionic surfactant is at least one ofpolysorbate 80 (TWEEN® 80), polyoxyl 15 hydroxystearate (KOLLIPHOR® HS15), polyoxyethylene castor oil derivatives, and other non-ionicsurfactants, or combination thereof.

Preferably, the second non-ionic surfactant is at least one ofKOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP), KOLLIPHOR® RH 40 (polyoxyl 40 hydrogenated castor oil, formerlyknown as CREMOPHOR® RH 40), and other polyoxyethylene castor oilderivatives, or combination thereof.

Preferably, the second lipophilic drug is at least one of quercetin,synephrine, puerarin, resveratrol, and any other lipophilic drug exceptcurcuminoid, or combination thereof.

Preferably, the weight ratio of the curcuminoid to the second lipophilicdrug is 30:1-1:10.

Preferably, the weight ratio of the curcuminoid to the lipophilic drug20:1-1:8.

Preferably, the pharmaceutically acceptable aqueous solution furthercomprises a hydrophilic drug.

Preferably, the hydrophilic drug is at least one of green tea extract,epigallocatechin gallate, epicatechin, epicatechin gallate,epigallocatechin, gallocatechin gallate, gallocatechin, catechingallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine,L-carnitine, synephrine, chlorogenic acid, and other hydrophilic drugs,or combination thereof.

Preferably, the weight ratio of the curcuminoid to the hydrophilic drugis 30:1 to 1:10.

Preferably, the weight ratio of the curcuminoid to the hydrophilic drugis 20:1 to 1:8.

Preferably, the formulation of the drug is subcutaneous injectionformulation, subcutaneous fat layer injection formulation, solution forimplanted infusion, cream formulation, patch formulation, or otherskin-absorption delivery systems.

Preferably, the drug is to be administered at an administration site toreduce the subcutaneous fat at the administration site.

Preferably, the formulation of the drug is subcutaneous injectionformulation or subcutaneous fat layer injection formulation, and theadministered dosage of the drug to be injected at the local site is0.02-20 mg/cm2.

The formulation of the drug is subcutaneous injection formulation orsubcutaneous fat layer injection formulation, and the administereddosage of the drug to be injected is 0.04-16 mg/cm′.

Preferably, the formulation of the drug is subcutaneous injectionformulation or subcutaneous fat layer injection formulation, and theadministered dosage of the drug to be injected is 0.01-40 mg/kg.

Preferably, the formulation of the drug is subcutaneous injectionformulation or subcutaneous fat layer injection formulation, and theadministered dosage of the drug to be injected is 0.1-20 mg/kg.

Preferably, the dosing frequency of the drug to be administered at theadministration site is 1-12 times every other day to every 30 days.

Preferably, the dosing frequency of the drug to be administered at theadministration site is 1-6 times every other day to every 30 days.

Preferably, the curcuminoid is curcumin.

Preferably, the pharmaceutical composition further comprises at leastone of a cosolvent, a suspending agent, and an oil phase excipient, orcombination thereof.

Preferably, the microstructure is co-formed by the non-ionic surfactantand at least one of the oil phase excipient and cosolvent.

The present invention further provides a use of a pharmaceuticalcomposition in preparing a drug or a subcutaneous injection formulationfor reducing weight; the pharmaceutical composition comprises:

-   -   drug-containing micelles; and    -   curcuminoid encapsulated in said drug-containing micelles;        wherein, the drug-containing micelle is a microstructure formed        by a pharmaceutically acceptable non-ionic surfactant, and the        hydrophilic-lipophilic balance value (HLB value) of the        non-ionic surfactant is greater than 10.

Preferably, the pharmaceutical composition further comprises apharmaceutically acceptable aqueous solution, and said drug-containingmicelles are evenly distributed in said pharmaceutically acceptableaqueous solution.

Preferably, the curcuminoid is curcumin.

Preferably, the pharmaceutical composition further comprises secondlipophilic drug-containing micelles, and the second lipophilicdrug-containing micelles are evenly distributed in the pharmaceuticallyacceptable aqueous solution; wherein, the second lipophilicdrug-containing micelle is a second microstructure formed by a secondnon-ionic surfactant, a second lipophilic drug is encapsulated in saidsecond lipophilic drug-containing micelles, and the second lipophilicdrug is at least one of quercetin, synephrine, puerarin, resveratrol,and any other lipophilic drug except curcuminoid, or combinationthereof.

Preferably, the pharmaceutically acceptable aqueous solution furthercomprises a hydrophilic drug, and the hydrophilic drug is at least oneof green tea extract, epigallocatechin gallate, epicatechin, epicatechingallate, epigallocatechin, gallocatechin gallate, gallocatechin,catechin gallate, catechin, epigallocatechin gallate (EGCG), caffeine,carnitine, L-carnitine, synephrine, chlorogenic acid, and otherhydrophilic drugs, or combination thereof.

Preferably, the formulation of the drug is subcutaneous injectionformulation, intravenous injection formulation, or subcutaneous fatlayer injection formulation, and the administered dosage of the drug tobe injected is 0.2-16 mg/cm2.

Preferably, the formulation of the drug is subcutaneous injectionformulation, intravenous injection formulation, or subcutaneous fatlayer injection formulation, and the administered dosage of the drug tobe injected is 0.4-8 mg/cm2.

Preferably, the formulation of the drug is subcutaneous injectionformulation, intravenous injection formulation, or subcutaneous fatlayer injection formulation, and the administered dosage of the drug tobe administered is 0.4-40 mg/kg.

Preferably, the formulation of the drug is subcutaneous injectionformulation, intravenous injection formulation, or subcutaneous fatlayer injection formulation, and the administered dosage of the drug tobe injected is 0.8-20 mg/kg.

Preferably, the dosing frequency of the drug to be administered at theadministered site is 3-60 times every other day to every 20 days.

Preferably, the dosing frequency of the drug to be administered at theadministered site is 6-42 times every other day to every 14 days.

Preferably, the pharmaceutical composition further comprises at leastone of a cosolvent, a suspending agent, and an oil phase excipient, orcombination thereof.

Preferably, the microstructure is co-formed by the non-ionic surfactantand at least one of the oil phase excipient and cosolvent.

The present invention provides a method for reducing subcutaneous fat ata local site of a subject, comprising a step of administering apharmaceutical composition at the local site of the subject, wherein,the pharmaceutical composition comprises:

-   -   drug-containing micelles; and    -   curcuminoid encapsulated in said drug-containing micelles;        wherein, the drug-containing micelle is a microstructure formed        by a pharmaceutically acceptable non-ionic surfactant, and the        hydrophilic-lipophilic balance value (HLB value) of the        non-ionic surfactant is greater than 10.

Preferably, the step is to administer the pharmaceutical composition atthe local site of the subject with a subcutaneous injection formulation,a subcutaneous fat layer injection formulation, a solution for implantedinfusion, cream formulation, a patch formulation, or otherskin-absorption delivery systems.

Preferably, the administered dosage of the pharmaceutical composition tobe injected at the local site of the subject is 0.02-20 mg/cm2

Preferably, the administered dosage of the pharmaceutical composition tobe injected at the local site of the subject is 0.01-40 mg/kg

Preferably, the dosing frequency of the pharmaceutical composition to beadministered at the local site of the subject is 1-12 times every otherday to every 30 days.

The present invention provides a method for reducing body weight of asubject, comprising a step of administering a pharmaceutical compositionto the subject, wherein, the pharmaceutical composition comprises:

-   -   drug-containing micelles; and    -   curcuminoid encapsulated in said drug-containing micelles;        wherein, the drug-containing micelle is a microstructure formed        by a pharmaceutically acceptable non-ionic surfactant, and the        hydrophilic-lipophilic balance value (HLB value) of the        non-ionic surfactant is greater than 10.

Preferably, the step is to administer the pharmaceutical composition atthe local site of the subject with a subcutaneous injection formulation,an intravenous injection formulation, or a subcutaneous fat layerinjection formulation.

Preferably, the administered dosage of the pharmaceutical composition tobe injected at the local site of the subject is 0.2-16 mg/cm2.

Preferably, the administered dosage of the pharmaceutical composition tobe injected at the local site of the subject is 0.4-40 mg/kg.

Preferably, the dosing frequency of the pharmaceutical composition to beadministered at the local site of the subject is 3-60 times every otherday to every 20 days.

Preferably, the step is to administer the pharmaceutical composition atthe local site of the subject with a subcutaneous injection formulation,a subcutaneous fat layer injection formulation, an intravenous injectionformulation, a solution for implanted infusion, a cream, a patch, orother skin-absorption delivery systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: A bar graph showing the effects of turmeric extract on theamount of subcutaneous fat of rats by way of oral administration

FIG. 1B: A bar graph showing the effects of turmeric extract on totalbody weight gain of rats by way of oral administration

FIG. 2A: A bar graph showing the effects of curcumin subcutaneousinjection formulations prepared with different excipients on the amountof localized subcutaneous fat of rats

FIG. 2B: A bar graph showing the effects of curcumin subcutaneousinjection formulations prepared with different excipients on total bodyweight gain of rats

FIG. 3: A bar graph showing the effects of micelles on the amount oflocalized subcutaneous fat of rats

FIG. 4: A bar graph showing the effects of micelles on total body weightgain of rats

FIG. 5: A bar graph showing the effects of resveratrol subcutaneousinjection formulation without excipient on the amount of localizedsubcutaneous fat of rats

FIG. 6: A bar graph showing the effects of curcumin-resveratrol complexsubcutaneous injection formulations prepared with different excipientson the amount of localized subcutaneous fat of rats

FIG. 7: A bar graph showing the effects of curcumin simple, resveratrolsimple, and curcumin-resveratrol complex subcutaneous injectionformulations comprising micelles on the amount of localized subcutaneousfat of rats

FIG. 8A: A bar graph showing the effects of dosing frequency ofcurcumin-resveratrol complex pharmaceutical composition on the amount oflocalized subcutaneous fat of rats

FIG. 8B: A bar graph showing the effects of dosing frequency ofcurcumin-resveratrol complex pharmaceutical composition on total bodyweight gain of rats

FIG. 9: A bar graph showing the effects of administered dosage ofcurcumin-resveratrol complex pharmaceutical composition on the amount oflocalized subcutaneous fat of rats.

FIG. 10: The effects of curcumin-other lipophilic drug complexpharmaceutical compositions on the apoptosis of mature adipocytes

FIG. 11: A bar graph showing the effects of green tea extractsubcutaneous injection formulation without excipient on the amount oflocalized subcutaneous fat of rats

FIG. 12A: A bar graph showing the effects of dosing frequency ofcurcumin-green tea extract complex pharmaceutical composition on theamount of localized subcutaneous fat of rats

FIG. 12B: A bar graph showing the effects of dosing frequency ofcurcumin-green tea extract complex pharmaceutical composition on totalbody weight gain of rats

FIG. 13: The effects of curcumin-other hydrophilic drug complexpharmaceutical compositions on the apoptosis of mature adipocytes

DETAILED DESCRIPTION OF THE INVENTION Experiment 1: The Effects ofOrally Administered Turmeric Extract on the Amount of the SubcutaneousFat and the Body Weight of Rats

The turmeric extract oral liquid was prepared as follows: An appropriateamount of sterile water for injection was added to an appropriate amountof turmeric extract and mixed well to obtain the turmeric extract oralliquid.

Seven-week-old male Sprague-Dawley rats were used for the experiment.First, 12 rats were fed with high-fat diet (Research Diets, Inc.; Cat#D12492) to induce the accumulation of subcutaneous fat until each ratweighed 330±10 g, and the rats were randomly assigned into two groups,which were a high-fat diet control group, a turmeric extract oraladministration group, with 6 rats in each group such that there was nostatistical difference in the body weight between groups. The bodyweight of each rat was recorded and defined as the “pre-experimentalbody weight” of each rat. Then, drugs were administered as follows:

Turmeric extract oral administration group: the rats were fed withhigh-fat diet daily and administered with turmeric extract oral liquidvia oral gavage, and the administered dosage of turmeric extract was 100mg/kg/day, and the rats were fed for 20 consecutive days. Theconcentration of curcumin in the turmeric extract oral liquid is 95% (wt%).

High-fat diet control: rats were fed with high-fat diet daily but werenot fed with turmeric extract.

Changes of the body weight were recorded daily during the period of theexperiment, and water and food consumption was recorded weekly. The ratswere fasted on day 20 and euthanized on day 21.

The body weight of each rat was recorded and defined as the“post-experimental body weight” of each rat. The “total body weightgain” of each rat was obtained by subtracting its “pre-experimental bodyweight” from its “post-experimental body weight”. Finally, the bilaterallower inguinal subcutaneous fat pads were dissected and weighed, and theamount of the inguinal fat of each group was calculated. The data werepresented as mean t SD and statistically analyzed. The statisticalresults were shown as symbols or letters, wherein different symbols orletters indicates statistically significant difference (p<0.05), andidentical symbols or letters indicates no statistical significance(p>0.05).

Please refer to FIG. 1A and FIG. 1B. FIG. 1A is a bar graph showing theeffect of turmeric extract on the amount of subcutaneous fat of rats viaoral administration. Wherein, said inguinal subcutaneous fat is thetotal amount of bilateral inguinal subcutaneous fat. FIG. 1B is a bargraph showing the effects of turmeric extract on total body weight gainof rats via oral administration.

As shown in FIG. 1A, the inguinal subcutaneous fat of the rats inhigh-fat diet control group is 6.4±1.5 g, and the inguinal subcutaneousfat of the rats in turmeric extract oral administration group is 6.1±0.8g. and there is no statistical significance between groups (p>0.05).This demonstrated that oral administration of turmeric extract cannotreduce localized fat.

As shown in FIG. 1B, the total body weight gain of the rats in high-fatdiet control group is 135±12 g, and the total body weight gain of therats in high-fat diet control group is 142±10 g, and there was nostatistical significance between groups (p>0.05). This demonstrated thatoral administration of turmeric extract cannot reduce the body weight.

The above experiments demonstrated that orally administrated turmericextract cannot reduce the localized fat nor can it reduce the bodyweight. In order to solve this problem, the inventor conducted furtherstudies to develop the pharmaceutical composition comprising curcuminand the subcutaneous injection formulation thereof.

Experiment 2: The Effects of Curcumin Subcutaneous Injection Formulationon the Amount of Subcutaneous Fat and the Body Weight of Rats

A curcumin normal saline solution, a curcumin PEG solution, and acurcumin ELP solution were prepared as follows:

Preparation of the Curcumin Normal Saline Solution:

-   -   450 mg of curcumin was mixed with an appropriate amount of        normal saline for injection to a total volume of 90 mL. The        solution was mixed well to completely dissolve curcumin to        obtain the curcumin normal saline solution, and the        concentration of curcumin in said curcumin normal saline        solution was 5 mg/mL.

Preparation of the Curcumin PEG Solution.

-   -   15 g of polyethylene glycol 400 (PEG 400) and 15 g of glycerol        were mixed with an appropriate amount of normal saline for        injection to a total volume of 100 mL. The solution was mixed        well to completely dissolve PEG 400 and glycerol to obtain a PEG        and glycerol mixture. 450 mg of curcumin was mixed with an        appropriate amount of the PEG and glycerol mixture to a total        volume of 90 mL. The solution was mixed well to completely        dissolve curcumin to obtain the curcumin PEG solution. The        concentration of curcumin in said curcumin PEG solution was 5        mg/mL.

Preparation of the Curcumin ELP Solution:

-   -   450 mg of curcumin was mixed with 80-A40 mL of dichloromethane,        and stirred at 150-500 rpm at room temperature until curcumin        dissolved completely. 18 g of KOLLIPHOR® ELP (polyoxyl-35-castor        oil, formerly known as CREMOPHOR® ELP, abbreviated as ELP) was        added to the solution and stirred well at 100-300 rpm to        volatilize dichloromethane. Once dichloromethane volatilized        completely, normal saline for injection was slowly added to a        total volume of 90 mL, and the solution was mixed well to obtain        the curcumin ELP solution. In said curcumin ELP solution, the        concentration of curcumin was 5 mg/mL, the ELP concentration was        approximately 20% (wt %), and the weight ratio of curcumin to        ELP was 1:40.

Six-week-old male Sprague-Dawley rats were used for the experiment.First, 20 rats were fed with high-fat diet (Research Diets. Inc.; Cat#D12492) to induce the accumulation of subcutaneous fat. Feeding wascontinued until each rat weighed 330±10 g, and the rats were randomlyassigned into four groups, which were a control group, a normal salinegroup, a PEG group, and an ELP group, with 5 rats in each group suchthat there was no statistical difference in the body weight betweengroups. The body weight of each rat was recorded and defined as the“pre-experimental body weight” of each rat. Then, drugs wereadministered as follows:

The curcumin normal saline, the curcumin PEG solution, and the curcuminELP solution were injected to the inguinal subcutaneous fat pads of ratsin the normal saline group, the PEG group, and the ELP group,respectively. Each injection volume was 4 mL per kilogram of body weight(4 mL/kg), such that each injected dosage was 20 mg of curcumin perkilogram of body weight (20 mg/kg; 4 mL/kg×5 mg/mL=20 mg/kg). Rats inthe control group were injected with the same volume of normal saline inthe same manner described above.

The injection sites mentioned above were the lower inguinal fat pads ofrats. Bilateral injections were administered evenly once a day on day 1,2, 3, and 4 of the experiment. The rats were fed with high-fat diet forthe entire duration of the experiment. Their weight changes wererecorded daily, and food and water consumption was recorded weekly. Theexperiment lasted for 14 days, and the rats were euthanized by CO2 onday 15.

The body weight of each rat was recorded and defined as the“post-experimental body weight” of each rat. The “total body weightgain” of each rat was obtained by subtracting its “pre-experimental bodyweight” from its “post-experimental body weight”. The “relative weightgain” was obtained by dividing the total body weight gain of each groupby the total body weight gain of the control group.

The bilateral lower inguinal subcutaneous fat pads of rats weredissected and weighed, and the weights of the bilateral lower inguinalsubcutaneous fat pads were summed to calculate the amount of loweringuinal subcutaneous fat. The amount of lower inguinal subcutaneous fatof each group was divided by the amount of lower inguinal subcutaneousfat of the control group to obtain the “relative weight of the loweringuinal subcutaneous fat”.

The data were presented as mean+SD and analyzed by one-way ANOVA.Statistical results were shown as symbols or letters. Different symbolsor letters indicates statistically significant difference (p<0.05), andidentical symbols or letters indicates no statistically significantdifference (p>0.05).

Please refer to FIG. 2A and FIG. 2B. FIG. 2A is a bar graph showing theeffects of curcumin subcutaneous injection formulations prepared withdifferent excipients on the amount of localized subcutaneous fat ofrats. FIG. 2B is a bar graph showing the effects of curcuminsubcutaneous injection formulations prepared with different excipientson total body weight gain of rats.

As shown in FIG. 2A, the relative weight of the lower inguinalsubcutaneous fat of rats in the control group was 100±27.6%, therelative weight of the lower inguinal subcutaneous fat of rats in thenormal saline group was 99.8±8.0%, the relative weight of the loweringuinal subcutaneous fat of rats in the PEG group was 93.6±5.8%, andthe relative weight of the lower inguinal subcutaneous fat of rats inthe ELP group was 62.8±20.5%. There was no significant difference in therelative weight of the lower inguinal subcutaneous fat between thenormal saline and the control group, suggesting that direct injection ofcurcumin to the subcutaneous fat layer of the administration site cannotreduce the fat at the administration site (localized fat). There was nosignificant difference in the relative weight of the lower inguinalsubcutaneous fat between the PEG group and the control group; therelative weight of the lower inguinal subcutaneous fat of the ELP groupwas significantly different (p<0.05) from that of the control group, andthe relative weight of the lower inguinal subcutaneous fat of rats inthe ELP group was reduced by 37.2%,

As shown in FIG. 2B, the relative weight gain of rats in the controlgroup was 100.0±30.8%, the relative weight gain of rats in the normalsaline group was 110.0±18.7%, the relative weight gain of rats in thePEG group was 112.5±20.7%, and the relative weight gain of rats in theELP group was 87.1±33.1%. There was no statistical significance betweenthe four groups (p>0.05), but the body weight of rats in the ELP groupwas 12.9% less than the body weight of rats in the control group,indicating a trend of body weight loss in rats of the ELP group.

The experiments above demonstrated that direct injection of curcumin tothe subcutaneous fat layer of the administration site cannot reduce thefat at the administration site (localized fat), nor can it reduce thebody weight. Direct injection of the curcumin composition comprising theexcipient PEG (a commonly used suspending agent) to the subcutaneous fatlayer of administration site cannot reduce the fat at the administrationsite (localized fat), nor can reduce body weight; however, injection ofthe curcumin composition comprising the non-ionic surfactant ELP to thesubcutaneous fat layer of the administration site not only can reducethe fat at the administration site (localized fat), but can also lead toa trend of body weight loss. Thus, it is necessary to furtherinvestigate whether the curcumin mixture has to comprise non-ionicsurfactants to reduce the fat at the administration site (localized fat)and to reduce body weight.

Further analysis showed that there were no micelles in the administeredcurcumin PEG solution mentioned above, but there were micelles in thecurcumin ELP solution, and curcumin was encapsulated in the micellesformed by ELP. Thus, it is necessary to further investigate the effectof micelles on reducing localized fat and reducing body weight.

Experiment 3: The Effects of Curcumin Simple Composition SubcutaneousInjection Formulation Comprising Non-Ionic Surfactants on the Amount ofSubcutaneous Fat and the Body Weight of Rats

A curcumin partial micellar formulation, a curcumin HS-15 partialmicellar formulation, a curcumin ELP micellar formulation, and acurcumin HS-15 micellar formulation were prepared as follows:

Preparation of the curcumin ELP partial micellar formulation: 20 g ofKOLLIPHCOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP, abbreviated as ELP) was mixed with an appropriate amount of normalsaline for injection to a total weight of 100 g. The solution was mixedwell to completely dissolve ELP to obtain a 20% ELP solution. 400 mg ofcurcumin was mixed with an appropriate amount of the 20% ELP solution toa total weight of 80 g. The solution was mixed well to completelydissolve curcumin to obtain the curcumin ELP partial micellarformulation. The concentration of curcumin in said curcumin ELP partialmicellar formulation was approximately 5 mg/mL, the concentration of ELPwas approximately 20%, and the weight ratio of curcumin to ELP wasapproximately 1:40.

Preparation of the curcumin HS-15 partial micellar formulation: 20 g ofKOLLIPHOR® HS-15 (polyoxyl 15 hydroxystearate, abbreviated as HS-15) wasmixed with an appropriate amount of normal saline for injection to atotal weight of 100 g. The solution was mixed well to completelydissolve HS-15 to obtain a 20% HS-15 solution. 400 mg of curcumin wasmixed with an appropriate amount of the 20% HS-15 solution to a totalweight of 80 g. The solution was mixed well to completely dissolvecurcumin to obtain the curcumin HS-15 partial micellar formulation. Theconcentration of curcumin in said curcumin HS-15 partial micellarformulation was approximately 5 mg/mL, the concentration of ELP wasapproximately 20% (wt %), and the weight ratio of curcumin to HS-15 wasapproximately 1:40.

Preparation of the Curcumin ELP Micellar Formulation: Same as thePreparation of the Curcumin ELP Solution in Experiment 2.

Preparation of the curcumin HS-15 micellar formulation: 500 mg ofcurcumin was mixed with 80-140 mL of dichloromethane and stirred at150-500 rpm at room temperature until curcumin dissolved completely. 20g of KOLLIPHOR® HS-15 (polyoxyl 15 hydroxystearate, abbreviated asHS-15) was added and stirred at 100-300 rpm to volatilizedichloromethane. Once dichloromethane volatilized completely, normalsaline for injection was slowly added to a total volume of 100 g. Thesolution was mixed well to form drug-containing micelles to obtain thecurcumin HS-15 micellar formulation. The concentration of curcumin insaid curcumin HS-15 micellar formulation was approximately 5 mg/mL, theconcentration of HS-15 was approximately 20%, and the weight ratio ofcurcumin to HS-15 was approximately 1:40.

The curcumin ELP partial micellar formulation, the curcumin HS-15partial micellar formulation, the curcumin ELP micellar formulation, andthe curcumin HS-15 micellar formulation were analyzed by a particle sizeanalyzer to determine if micelles were present, and the diameters of themicelles were measured.

The results showed that both the curcumin ELP partial micellarformulation and the curcumin HS-15 partial micellar formulation had drugprecipitates (curcumin precipitates), and had a lower number ofdrug-containing micelles. On the contrary, the curcumin ELP micellarformulation and the curcumin HS-15 micellar formulation were clearwithout any stratification, and had a higher number of drug-containingmicelles.

In addition, the particle diameters of the micelles in the curcumin ELPpartial micellar formulation, the curcumin HS-15 partial micellarformulation, the curcumin ELP micellar formulation, and the curcuminHS-15 micellar formulation were 13.16±0.18 nm. 13.18±1.45 nm. 12.43±0.40nm, and 11.46±0.41 nm, respectively, and the PDI values were 0.22±0.03,0.18±0.05, 0.28±0.05, and 0.18±0.04, respectively.

The results indicated that although the curcumin ELP partial micellarformulation and the curcumin HS-15 partial micellar formulation both haddrug precipitates (curcumin precipitates), their supernates stillcontained micelles (diameter <250 nm and PDI value <0.4). Therefore, thecurcumin ELP partial micellar formulation, the curcumin HS-15 partialmicellar formulation, the curcumin ELP micellar formulation, and thecurcumin HS-15 micellar formulation are all pharmaceutical compositionsof the present invention.

Six-week-old male Sprague-Dawley rats were used for the experiment.First, 20 rats were fed with high-fat diet (Research Diets, Inc.; Cat#D12492) to induce the accumulation of subcutaneous fat. Feeding wascontinued until each rat weighed 330±10 g, and the rats were randomlyassigned into 5 groups, which were a control group, an ELP partialmicellar group, an HS-15 partial micellar group, an ELP micellar group,and an HS-15 micellar group, with 4 rats in each group such that therewas no statistical difference in the body weight between groups. Thebody weight of each rat was recorded and defined as the“pre-experimental body weight” of each rat. Then, drugs wereadministered as follows:

The curcumin ELP partial micellar formulation, the curcumin HS-15partial micellar formulation, the curcumin ELP micellar formulation, andthe curcumin HS-15 micellar formulation were each prepared and mixedwell (to evenly suspend the precipitates in the partial micellarformulations), and were injected into the lower inguinal subcutaneousfat layer of rats in the ELP partial micellar group, the HS-15 partialmicellar group, the ELP micellar group, and the HS-15 micellar group,respectively. Each injection volume was 4 mL per kilogram of body weight(4 mg/kg), such that each injected dosage was 20 mg of curcumin perkilogram of body weight (20 mg/kg; 4 mL/kg×5 mg/mL=20 mg/kg). Rats inthe control group were injected with the same volume of normal saline inthe same manner described above.

The injection sites mentioned above were the lower inguinal fat pads ofrats. Bilateral injections were administered evenly once a day on day 1,2, 3, 4, 5, and 6 of the experiment. The rats were fed with high-fatdiet for the entire duration of the experiment. Their weight changeswere recorded daily, and food and water consumption was recorded weekly.The experiment lasted for 14 days, and the rats were euthanized by CO2on day 15.

The body weight of each rat was recorded and defined as the“post-experimental body weight” of each rat. The “total body weightgain” of each rat was obtained by subtracting its “pre-experimental bodyweight” from its “post-experimental body weight”. The “relative weightgain” was obtained by dividing the total body weight gain of each groupby the total body weight gain of the control group.

The bilateral lower inguinal subcutaneous fat pads of rats weredissected and weighed, and the weights of the bilateral lower inguinalsubcutaneous fat pads were summed to calculate the amount of loweringuinal subcutaneous fat. The amount of lower inguinal subcutaneous fatof each group was divided by the amount of lower inguinal subcutaneousfat of the control group to obtain the “relative weight of the loweringuinal subcutaneous fat”.

The data were presented as mean t SD and analyzed by one-way ANOVA.Statistical results were shown as symbols or letters. Different symbolsor letters indicates statistically significant difference (p<0.05), andidentical symbols or letters indicates no statistically significantdifference (p>0.05).

Based on the formulation preparation methods and the results of particlesize analysis above, the concentration of ELP and the concentration ofcurcumin in the curcumin ELP partial micellar formulation were identicalto those of the curcumin ELP micellar formulation, only the number ofdrug-containing micelles differed. Thus, comparing to the control group,if the curcumin ELP partial micellar formulation cannot significantlyreduce the localized fat at the administration site, but the curcuminELP micellar formulation can significantly reduce the localized fat atthe administration site, this indicates that formation ofdrug-containing micelles is the critical factor for curcumincompositions to significantly reduce the localized fat at theadministration site.

Similarly, the concentration of HS-15 and the concentration of curcuminin the curcumin HS-15 partial micellar formulation were identical tothose of the curcumin HS-15 micellar formulation, only the number ofdrug-containing micelles differed. Thus, comparing to the control group,if the curcumin HS-15 partial micellar formulation cannot significantlyreduce the localized fat at the administration site, but the curcuminHS-15 micellar formulation can significantly reduce the localized fat atthe administration site, this indicates that formation ofdrug-containing micelles is the critical factor for curcumincompositions to significantly reduce the localized fat at theadministration site.

On the other hand, the concentration of ELP and the concentration ofcurcumin in the curcumin ELP partial micellar formulation were identicalto those of the curcumin ELP micellar formulation, only the number ofdrug-containing micelles differed. Thus, comparing to the control group,if the curcumin ELP partial micellar formulation cannot significantlyreduce the body weight, but the curcumin ELP micellar formulation cansignificantly reduce the body weight, this indicates that formation ofdrug-containing micelles is the critical factor for curcumincompositions to significantly reduce body weight.

Please refer to FIG. 3. FIG. 3 is a bar graph showing the effect ofmicelles on the amount of localized subcutaneous fat of rats. In FIG. 3,the y-axis is the relative weight of the lower inguinal subcutaneous fat(%), and the x-axis from left to right is the control group, the HS-15partial micellar group, the HS-15 micellar group, the ELP partialmicellar group, and the ELP micellar group, respectively.

As shown in FIG. 3, the relative weight of the lower inguinalsubcutaneous fat of rats in the control group was 100.0±26.4%, therelative weight of the lower inguinal subcutaneous fat of rats in theHS-15 partial micellar group was 74.7±10.1%, the relative weight of thelower inguinal subcutaneous fat of rats in the HS-15 micellar group was67.6±8.6%, the relative weight of the lower inguinal subcutaneous fat ofrats in the ELP partial micellar group was 71.8±22.9%, and the relativeweight of the lower inguinal subcutaneous fat of rats in the ELPmicellar group was 65.0±7.2%.

Comparing to rats in the control group, rats in the HS-15 partialmicellar group and the ELP partial micellar group showed a trend ofreduction in their relative weights of the lower inguinal subcutaneousfat, but the difference did not reach statistical significance. Therelative weights of lower inguinal subcutaneous fat of rats in the HS-15micellar group and the ELP micellar group decreased significantly(p<0.05) by 32.4% and 35%, respectively.

The data above demonstrated that although the concentration of non-ionicsurfactant and the concentration of curcumin in the partial micellarformations were identical to those of the micellar formations, and thatthe partial micellar formations contain some micelles, the partialmicellar formations can only promote a trend of reduction of thelocalized fat but cannot significantly reduce the localized fat. On thecontrary, the micellar formations with numerous micelles cansignificantly reduce the localized fat.

This demonstrated that formation of drug-containing micelles is acritical factor for curcumin compositions to significantly reduce thelocalized fat at the administration site. That is, curcumin compositionscomprise few drug-containing micelles can induce a trend of localizedfat reduction, and curcumin compositions with numerous drug-containingmicelles can significantly reduce the localized fat.

Please refer to FIG. 4. FIG. 4 is a bar graph showing the effects ofmicelles on total body weight gain of rats. In FIG. 4, the y-axis isrelative weight gain (%), and the x-axis from left to right is thecontrol group, the HS-15 partial micellar group, the HS-15 micellargroup, the ELP partial micellar group, and the ELP micellar group,respectively.

FIG. 4 shows that the relatively weight gain of rats in the controlgroup is 100.0±20.6%, the relatively weight gain of rats in the HS-15partial micellar group is 100.0±17.3%, the relatively weight gain ofrats in the HS-15 micellar group is 96.4±18.5%, the relatively weightgain of rats in the ELP partial micellar group is 73.8±11.2%, and therelatively weight gain of rats in the ELP micellar group is 54.8±14.3%.

Comparing to rats in the control group, rats in the ELP partial micellargroup showed a trend of reduction in their relative weight gain but thedifference did not reach statistical significance (p>0.05). The relativeweight gain of rats in the ELP micellar group was reduced by 45.2%, andwas significantly different from that of the control group (p<0.05).

The data above showed that although the concentration of non-ionicsurfactant and the concentration of curcumin in the partial micellarformations were identical to those of the micellar formations, and thatthe partial micellar formations contained some micelles, the partialmicellar formations can only induce a trend of reduction of the bodyweight but not significantly reduce the body weight. On the contrary,the micellar formations with numerous micelles can significantly reducethe body weight.

This demonstrated that formation of drug-containing micelles is acritical factor for curcumin compositions to significantly reduce thebody weight. That is, curcumin compositions with few drug-containingmicelles can promote a trend of body weight loss, and curcumincompositions with numerous drug-containing micelles can significantlyreduce the body weight.

Although the HS-15 micellar formulation did not significantly reduce thebody weight in this experiment, based on the experiences of theinventor, the HS-15 micellar formulation can also significantly reducethe body weight if dosing frequency or administered dosage is increased.Therefore, the non-ionic surfactant HS-15 should also be included in thescope of the present invention.

Experiment 4: Preparation of the Pharmaceutical Compositions of thePresent Invention

The experiments above demonstrated that using non-ionic surfactants toform micelles is the critical factor for curcumin compositions tosignificantly reduce localized fat. Therefore, the present inventionprovides a simple curcumin pharmaceutical composition for reducinglocalized fat, and its characteristics is that the curcumin simplepharmaceutical composition comprises drug-containing micelles.

Preparation of the Curcumin Simple Pharmaceutical Composition is asFollows:

-   -   (a) Mixing a first weight of curcumin with a solvent, and        stirring at 150-500 rpm at room temperature until curcumin        dissolves completely;    -   (b) Adding a second weight of a pharmaceutically acceptable        surfactant, and stirring well at 100-300 rpm to volatilize the        solvent. Wherein, the hydrophilic-lipophilic balance value (HLB        value) of the surfactant is greater than 10; and    -   (c) Once the solvent volatilizes completely, slowly adding a        third weight of a pharmaceutically acceptable aqueous solution        to obtain drug-containing micelles; and    -   (d) Filtering through a 0.2 um filter, and storing the filtered        solution comprising drug-containing micelles in dark and        refrigeration;

[Wherein, in step (c), the drug-containing micelle is a microstructureformed by the surfactant, and curcumin is encapsulated in saiddrug-containing micelle; the third weight is greater than or equal to 0g.

Preferably, the operating procedure of step (c) is: Once the solventvolatilizes completely, slowly adding the third weight of thepharmaceutically acceptable aqueous solution, and mixing well to formdrug-containing micelles.

Preferably, in step (a), the boiling point of the solvent is lower thanthat of pure water.

Preferably, in step (a), the solvent is a hydrophilic solvent.

Preferably, the hydrophilic solvent is at least one of methanol,ethanol, acetone, and other hydrophilic solvents, or combinationthereof.

Preferably, the solvent in step (a) is a lipophilic (hydrophobic)solvent.

Preferably, the lipophilic (hydrophobic) solvent is at least one ofether, benzene, chloroform, ethyl acetate, dichloromethane, hexane, andother lipophilic (hydrophobic) solvents, or combination thereof.

Preferably, in step (b), the surfactant is a non-ionic surfactant.

Preferably, the non-ionic surfactant is at least one of polysorbate 80(TWEEN® 80), polyoxyl 15 hydroxystearate (KOLLIPHOR® HS 15),polyoxyethylene castor oil derivatives, and other non-ionic surfactants,or combination thereof.

Preferably, the polyoxyethylene castor oil derivative is at least one ofKOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP), KOLLIPHOR® RH40 (polyoxyl 40 hydrogenated castor oil, formerlyknown as CREMOPHOR® RH 40), and other polyoxyethylene castor oilderivatives, or combination thereof.

Preferably, in steps (a) and (b), the weight ratio of the curcumin ofthe first weight and the surfactant of the second weight is 1:5 to1:500.

Preferably, in steps (a) and (b), the weight ratio of the curcumin ofthe first weight and the surfactant of the second weight is 1:20 to1:150.

Preferably, in steps (a) and (c), the weight ratio of the curcumin ofthe first weight and the pharmaceutically acceptable aqueous solution ofthe third weight is 1:400 to 3:50.

Preferably, in step (c), the pharmaceutically acceptable aqueoussolution is water for injection, aqueous solution for injection, ornormal saline.

Preferably, in step (c), the pharmaceutically acceptable aqueoussolution comprises a local anesthetic.

Preferably, in step (c), the pharmaceutically acceptable aqueoussolution comprises an antioxidant.

Experiment 5: Determination of the Quality of PharmaceuticalCompositions Experiment 5-1: Composition Analysis

Letting the pharmaceutical composition stand for at least 20 minutes. Ifthe composition does not have stratification, further analyzing it by aparticle analyzer.

Determining whether the pharmaceutical composition comprises micelles bya particle size analyzer. If the particle diameter of the pharmaceuticalcomposition, after being analyzed by a particle analyzer, is smallerthan 250 nm and the PDI value is less than 0.4, the solution of thepharmaceutical composition is deemed clear and transparent when observedby the naked eye, and the light beam can be observed when the solutionof the pharmaceutical composition is shined by a laser, then itindicates that the pharmaceutical composition comprises micelles.

If micelles are present in the pharmaceutical composition, the preparedpharmaceutical composition is the pharmaceutical composition forreducing localized fat in the present invention.

Preferably, if the pharmaceutical composition does not havestratification and does not contain precipitates after being let stand,the prepared pharmaceutical composition is the preferable pharmaceuticalcomposition of the present invention.

Experiment 5-2: Determination of the Stability of PharmaceuticalCompositions by Analyzing the Distribution of Particle Diameters

Using a particle size analyzer (purchased from Malvern) to determine thedistribution of particle diameters and the polydispersity index (PDI).If PDI is less than 0.4, it indicates that the stability ofpharmaceutical composition is good, that is, the micelles in thepharmaceutical composition can exist stably.

Experiment 5-3: Determination of the Stability of PharmaceuticalCompositions by Accelerated Stability Test

The storage condition of the pharmaceutical composition of the presentinvention is 2-8° C. In order to test the stability of thepharmaceutical compositions, the inventor placed the pharmaceuticalcompositions in an environment of relatively high temperature andrelatively high humidity (temperature 25° C.±2° C., relative humidity60%±5%) for accelerated stability test, observed how long the micellesin the pharmaceutical composition can stably exist in a condition ofrelatively high temperature, to reckon the shelf life of thepharmaceutical composition at 2-8° C. based on the accelerated stabilitytest equation, as detailed below.

If the pharmaceutical composition has a shelf life of n months at acondition of 25° C., then the shelf life of the pharmaceuticalcomposition at a condition of 5° C. is 2((25-5)11°) folds of n months.That is, the shelf life of the pharmaceutical composition at a conditionof 5° C. is 22 folds of n months, that is, 4 folds.

For example, if the shelf life of the pharmaceutical composition is 6months at a condition of 25° C., then the shelf life of thepharmaceutical composition at a condition of 5° C. is 24 months (6months×4 folds=24 months.)

Preferably, the pharmaceutical composition maintains at a state withoutprecipitation for at least 24 hours when it is subjected to acceleratedstability test at a condition of temperature of 25° C.±2° C., relativelyhumidity of 60%±5%, and in the absence of direct light.

Preferably, the pharmaceutical composition maintains at a state withoutprecipitation for at least 6 months when it is subjected to acceleratedstability test at a condition of temperature of 25° C.±2° C., relativelyhumidity of 60%±5%, and in the absence of direct light.

Preferably, the pharmaceutical composition maintains at a state withoutprecipitation for at least 24 months at a condition of temperature of2-8° C.

Experiment 6: Maximum Drug Loading of Drug-Containing Micelles Formed byDifferent Non-Ionic Surfactants

Because the maximum drug loading of drug-containing micelles directlyaffects injected volume, it has huge impacts on the volume of drug, sideeffects, and the burden that have to be tolerated by localizedsubcutaneous fat layer (e.g. the subcutaneous fat layer of the face) ina single administration. Thus, this experiment investigates the maximumdrug loading of drug-containing micelles formed by different non-ionicsurfactants to determine which non-ionic surfactant is the bestexcipient for preparing the pharmaceutical compositions of the presentinvention.

Four non-ionic surfactants were selected for this experiment. The fournon-ionic surfactants were KOLLIPHOR® ELP (polyoxyl-35-castor oil,formerly known as CREMOPHOR® ELP, abbreviated as ELP). KOLLIPHOR® HS-15(polyoxyl 15 hydroxystearate, abbreviated as HS-15). KOLLIPHOR® RH 40(polyoxyl 40 hydrogenated castor oil, abbreviated as RH 40), andpolysorbate 80 (also known as TWEEN® 80).

The experiment was divided into 4 groups, which were an ELP group, aHS-15 group, a RH40 group, and a TWEEN® 80 group.

Experimental Procedure:

-   -   (a) 2.0 g (an example of the first weight) of curcumin was mixed        with 300500 mL of dichloromethane, and stirred at 150500 rpm at        room temperature until curcumin dissolved completely.    -   (b) 18.0 g (an example of the second weight) of one of the        non-ionic surfactants mentioned above was added to the solution,        and stirred at 100300 rpm to volatilize dichloromethane; and    -   (c) A composition of 20 g in total was obtained after the        solvent volatilized completely; 2 g of the composition was        weighed out, and 8 g (an example of the third weight) of normal        saline for injection was added and mixed well to obtain a        composition to be tested. The concentration of curcumin in the        composition to be tested was 20 mg/g, and the concentration of        the non-ionic surfactant was 18%.

The compositions to be tested from the ELP group, the HS-15 group, theRH40 group, and the TWEEN® 80 group were let stand for at least 20minutes to observe if stratification occurs. If stratification occurs,it indicates that the concentration of curcumin is too high and willcause the micelles in the composition to be tested to burst. That is,the non-ionic surfactant cannot be used to prepare the pharmaceuticalcompositions of the present invention which comprise as high as 20 mg/gof curcumin.

The experimental results showed that the compositions to be tested inthe HS-15 group and the RH40 group had stratification, and only thecompositions to be tested from the ELP group and the TWEEN® 80 group didnot have stratification. Therefore, the maximum drug loading ofdrug-containing micelles formed by HS-15 and RH40 are both smaller than20 mg/g. The drug-containing micelles formed by ELP and TWEEN® 80 can beused to prepare pharmaceutical compositions with 20 mg/g of curcumin.

Because TWEEN® 80 is toxic, different national pharmacopoeias all limitthe injection concentration of TWEEN® 80 to less than 0.4% to avoidadverse effects or toxicity. Thus, the maximum drug loading of thedrug-containing micelles formed by TWEEN® 80 should be 0.44 mg/g.(Calculation: 20 mg/g×(0.4%/18%)=0.44 mg/g.)

In order to determine the maximum drug loading of ELP, the inventorfurther performed Experiment 6 and determined that the maximum drugloading of ELP is greater than or equal to 111 mg/g. (When the ratio ofcurcumin to ELP is 1:8, the prepared pharmaceutical composition contains111 mg/g of curcumin.)

The results above indicated that ELP is the best excipient to preparethe pharmaceutical compositions of the present invention. Theconcentration of curcumin can reach 111 mg/g in the pharmaceuticalcompositions prepared with ELP, while the concentration of curcumin isless than 20 mg/g in the pharmaceutical compositions prepared with othernon-ionic surfactants (please refer to Table 1).

In order to determine which of the non-ionic surfactants between HS-15and RH40 has the minimum drug loading, the inventor further used thosenon-ionic surfactants to prepare the pharmaceutical compositions of thepresent invention with 10 mg/g of curcumin. The results showed that ELP,HS-15, RH40, and TWEEN® 80 can all be used to prepare the pharmaceuticalcompositions of the present invention with 10 mg/g of curcumin, and saidpharmaceutical compositions of the present invention with 10 mg/g ofcurcumin were clear without stratification, and the measured particlediameters were 15.95±0.24 nm. 88.23±116.06 nm, 21.63±9.34 nm, 11.37±0.13nm, respectively, and the PDI values were 0.32±0.02, 0.48±0.27,0.26±0.09, 0.33±0.04, respectively.

Wherein, when HS-15 was used to prepare the pharmaceutical compositionof the present invention with 10 mg/g of curcumin, the PDI value of theprepared pharmaceutical composition was greater than 0.4, and it did notsatisfy the definition of the presence of micelles in the pharmaceuticalcomposition of the present invention (measured particle diameter issmaller than 250 nm, PDI value is less than 0.4, the solution of thepharmaceutical composition is deemed clear and transparent when it isobserved by the naked eye, and light beam can be observed when thesolution of the pharmaceutical composition is shined by a laser.)Therefore, among the non-ionic surfactants selected for this experiment.HS-15 has the minimum drug loading.

TABLE 1 Maximum drug load of drug-containing micelles formed bydifferent non-ionic surfactants Maximum drug load of the micellesMaximum tolerated dosage Group (mg/g) of micellar drug load ELPgroup >111 >111 HS-15 group <10 <10 RH40 <20; ≥10 <20; ≥10 TWEEN ® 80 ≥20; 0.44

Experiment 6: Preparation of Pharmaceutical Compositions with KOLLIPHOR®ELP (Polyoxyl-35-Castor Oil. Abbreviated as ELP)

In order to determine both of the appropriate ratio between curcumin andKOLLIPHOR® ELP (polyoxyl-35-castor oil, abbreviated as ELP) and themaximum drug loading when preparing the pharmaceutical compositions inthe present invention with ELP, various ratios between curcumin andKOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP, abbreviated as ELP) were used in this experiment to prepare aseries of pharmaceutical compositions of the present invention, and thestability analysis thereof were performed.

There were 9 groups in this experiment, that is, the first to the ninthgroup. The preparation of pharmaceutical composition in each group wasroughly the same as the experimental procedure in Experiment 5. The onlydifferences were the weight of curcumin (the first weight in step (a′)),the weight of ELP (the second weight in step (b′)), and the weight ofnormal saline for injection (the third weight in step (c′)). In thisexperiment, the guideline of adding all of the weight of curcumin (thefirst weight), the weight of ELP (the second weight), and the weight ofnormal saline for injection (the third weight) was shown in Table 2.

In this experiment, the ratios of curcumin to ELP (weight ratio) in thefirst group to the ninth group were 1:4, 1:5, 1:8, 1:10, 1:20, 1:40,1:100, 1:150, 1:500, respectively, and the final concentrations ofcurcumin in the pharmaceutical compositions prepared in the first to theninth group were 200 mg/g, 167 mg/g, 111 mg/g, 91 mg/g, 47.62 mg/g, 7.5mg/g, 3 mg/g, 2 mg/g, and 0.5 mg/g, respectively. That is, thepreparation method of pharmaceutical composition in the first to theninth group, the weight ratios of curcumin in step (a′) to ELP in step(b′) (the ratios of the first weight to the second weight) were 1:4,1:5, 1:8, 1:10, 1:20, 1:40, 1:100, 1:150, 1:500, respectively, and thatafter adding the third weight of normal saline for injection in step(c′), the final concentrations of curcumin in the preparedpharmaceutical compositions were 200 mg/g, 167 mg/g, 111 mg/g, 91 mg/g,47.62 mg/g, 7.5 mg/g, 3 mg/g, 2 mg/g, and 0.5 mg/g, respectively.Wherein, when the final concentration of drug was presented as mg/g, itindicated the number of milligrams of curcumin per gram ofpharmaceutical composition.

Particle size analyzer was utilized to determine if micelles werepresent in the pharmaceutical compositions, and the particle diameter ofthe micelles was measured.

To assess the stability of the pharmaceutical compositions, thedistribution of particle diameters and the polydispersity index (PDI)were measured by a particle size analyzer. The curcumin content in themicelles was analyzed by high performance liquid chromatography (HPLC;e.g., HPLC-UV) and defined as the “initial drug content”.

TABLE 2 A sample preparation chart for preparing pharmaceuticalcompositions with ELP Ratio of Final concentration of curcumin curcuminto ELP in the pharmaceutical Group (weight ratio) composition (mg/g) 11:4 200 2 1:5 167 3 1:8 111 4  1:10 91 5  1:20 47.62 6  1:40 7.5 7 1:100 3 8  1:150 2 9  1:500 0.5

The pharmaceutical compositions were subjected to accelerated stabilitytest to observe if stratification occurred when the pharmaceuticalcompositions were stored at high temperature storage condition (25±2°C.) for 3 months. The drug content in the micelles was determined byhigh performance liquid chromatography (HPLC; e.g., HPLC-UV), anddefined as the “drug content after accelerated stability test”. The“percentage of drug content” was calculated by dividing the “drugcontent after accelerated stability test” by the “initial drug content”.If the percentage of drug content is greater than or equal to 95%, itindicates that the stability of the pharmaceutical composition isexcellent.

Please refer to Table 3. Table 3 is the stability analysis result of thepharmaceutical compositions. Table 3 shows the presence of micelles inthe second to the ninth pharmaceutical compositions. Therefore,pharmaceutical compositions prepared with curcumin to ELP ratios of 1:5to 1:500 are all pharmaceutical compositions for reducing localized fatin the present invention.

In terms of stability, when the ratios of curcumin to ELP were 1:4 and1:5. PDI was greater than 0.4. When the ratios of curcumin to ELP were1:8 to 1:500. PDI was smaller than 0.4. Thus, in order to prepare thepharmaceutical composition for reducing localized fat in the presentinvention with better stability, the ratio of curcumin to ELP should beless than one-fifth (⅕). That is, in order to prepare the pharmaceuticalcomposition for reducing localized fat in the present invention withbetter stability, based on 1 weight unit defined as the weight ofcurcumin, the weight of ELP should be greater than 5 weight units.Preferably, based on 1 weight unit defined as the weight of curcumin,the weight of ELP is 8500 weight units. Preferably, based on 1 weightunit defined as the weight of curcumin, the weight of ELP is 20150weight units.

Based on the data in Table 3, when the pharmaceutical compositions inthe fifth to the eighth group were stored at 25° C. for 3 months, thepercentage of curcumin drug content in every sample was greater than 95%and did not show a significant trend of decrease comparing to theinitial drug content. This result indicates that the pharmaceuticalcompositions have excellent stability, and based on the equation ofaccelerated stability test, the pharmaceutical compositions can bestored at 2-8° C. in refrigeration for at least 24 months.

TABLE 3 Stability analysis of the pharmaceutical compositions Ratio ofAppearance Drug content curcumin Micelle after after to ELP particleaccelerated accelerated Group (weight ratio) diameter (nm) PDI stabilitytest stability test 1 1:4  772.5 ± 198.92 0.79 ± 0.36 2 1:5 153.97 ±40.17  0.41 ± 0.13 3 1:8 13.17 ± 0.21   0.2 ± 0.02 4  1:10 12.47 ± 0.23 0.17 ± 0.01 5  1:20 12.57 ± 0.12  0.137 ± 0.03  Clear without 103.82 ±2.07   stratification 6  1:40 11.59 ± 0.27  0.174 ± 0.0   Clear without100.78 ± 0.51   stratification 7  1:100 12.26 ± 0.12  0.096 ± 0.07 Clear without 100.62 ± 0.21   stratification 8  1:150 12.93 ± 0.29 0.197 ± 0.02  Clear⁻ without 102.45 ± 0.05   stratification 9  1:50012.66 ± 0.14  0.16 ± 0.01

In the table above, blank cells indicate that the contents were notanalyzed.

Experiment 7: The Effects of Curcumin-Resveratrol Complex SubcutaneousInjection Formulation on the Subcutaneous Fat of Rats Experiment 7-1:The Effects of Resveratrol Simple Subcutaneous Injection Formulation onthe Subcutaneous Fat of Rats

Preparation of the Resveratrol Subcutaneous Injection Formulation:

-   -   Resveratrol was mixed with an appropriate amount of normal        saline for injection to obtain the resveratrol subcutaneous        injection formulation.

Rats were assigned into a high-fat diet control group and a resveratrolgroup with 6 rats per group. The rats were fed in the same mannerdescribed in Experiment 2. The resveratrol subcutaneous injectionformulation was injected to the lower inguinal subcutaneous fat layer ofrats in the resveratrol group, and each injected dosage was 8 mg ofresveratrol per kilogram of body weight (8 mg/kg). Rats in the high-fatdiet control group were injected with the same volume of water forinjection in the same manner described above.

The injection sites mentioned above were the lower inguinal fat pads ofrats. Bilateral injections were administered evenly once a day on day 1,3, and 5 of the experiment. The rats were fed with high-fat diet for theentire duration of the experiment. Their weight changes were recordeddaily, and food and water consumption was recorded weekly. Theexperiment lasted for 20 days, and the rats were euthanized on day 21 byCO2.

Please refer to FIG. 5. FIG. 5 is a bar graph showing the effects ofresveratrol subcutaneous injection formulation without excipient on theamount of localized subcutaneous fat of rats

Results from FIG. 5 showed that the relative weight of the loweringuinal subcutaneous fat of rats in the high-fat diet control group was100.00±21.51%, and the relative weight of the lower inguinalsubcutaneous fat of rats in the resveratrol group was 111.59±11.288%.There was no significant difference in the relative weight of the loweringuinal subcutaneous fat between rats in the resveratrol group and ratsin the high-fat diet control group, indicating that a lipophilic plantextract-resveratrol composition without excipient cannot reduce the fatat the administration site (localized fat). Therefore, the inventorbelieved that directly mixing resveratrol and curcumin and injectinginto the subcutaneous fat may not be able to reduce localized fat, sothe inventor further investigated if excipients can improve the locallipolysis efficacy of a complex drug (resveratrol+curcumin).

Experiment 7-2: The Effect of Curcumin-Resveratrol Complex Formula withExcipient on the Subcutaneous Fat of Rats

A curcumin-resveratrol complex solution comprising formulation A and acurcumin-resveratrol complex solution comprising ELP were prepared asfollows:

Preparation of the Curcumin-Resveratrol Complex Solution ComprisingFormulation A:

-   -   0.05 g of resveratrol, 0.2 g of curcumin, and 2 g of mannitol        were grinded and mixed well to obtain a powder formulation. 0.05        g of carboxymethylcellulose (CMC) was mixed with 40 mL of        sterile water and heated to 60° C.-70° C. to dissolve        carboxymethylcellulose (CMC), and 0.055 g of polysorbate 80        (TWEEN® 80) was added and mixed well until it dissolved        completely. Water was added to a total volume of 50 mL to obtain        a liquid formulation. The liquid formulation was added into the        powder formulation and mixed well to obtain the        curcumin-resveratrol complex solution comprising formulation A.        Said formulation A was TWEEN® 80 with mannitol. Said        curcumin-resveratrol complex solution comprising formulation A        did not comprise micelles, the concentration of curcumin was 4        mg/mL, and the concentration of resveratrol was 1 mg/mL.

Preparation of the Curcumin-Resveratrol Complex Solution Comprising ELP:

-   -   0.2 g of resveratrol, 0.8 g of curcumin, and 150-200 mL of        dichloromethane were mixed together, and stirred at 150-500 rpm        at room temperature until curcumin dissolved completely. 40 g of        KOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as        CREMOPHOR® ELP, abbreviated as ELP) was added and stirred at        100-300 rpm to volatilize dichloromethane. Once dichloromethane        volatilized completely, normal saline for injection was slowly        added to a total volume of 200 mL. The solution was mixed well        to obtain a curcumin-resveratrol complex solution comprising        ELP. Said curcumin-resveratrol complex solution comprising ELP        comprised micelles, the concentration of KOLLIPHOR® ELP        (polyoxyl-35-castor oil, abbreviated as ELP) was approximately        20%, and the weight ratio of curcumin, resveratrol, and ELP was        4:1:200.

The rats were randomly assigned into 4 groups, which were a high-fatdiet control group, a low-dosage complex formula with formulation Agroup, a high-dosage complex formula with formulation A group, and alow-dosage complex formula with ELP group. The rats were fed in the samemanner described in Experiment 2.

The curcumin-resveratrol complex solution comprising formulation A wasinjected to the inguinal subcutaneous fat layer of rats in thelow-dosage complex formula with formulation A group, and each injectionvolume was 0.2 ml of the curcumin-resveratrol complex solutioncomprising formulation A per kilogram of body weight (0.2 mL/kg), suchthat each injected dosage was 1 mg of curcumin-resveratrol complexformula per kilogram of body weight (1 mg/kg); the curcumin-resveratrolcomplex solution comprising formulation A was injected to the inguinalsubcutaneous fat layer of rats in the high-dosage complex formula withformulation A group, and each injection volume was 1 mL of thecurcumin-resveratrol complex solution comprising formulation A perkilogram of body weight, such that each injected dosage was 5 mg ofcurcumin-resveratrol complex formula per kilogram of body weight (5mg/kg); the curcumin-resveratrol complex solution comprising ELP wasinjected to the inguinal subcutaneous fat layer of rats in thelow-dosage complex formula with ELP group, and each injection volume was0.2 mL of the curcumin-resveratrol complex solution comprising ELP perkilogram of body weight (0.2 mL/kg), such that each injected dosage was1 mg of curcumin-resveratrol complex formula per kilogram of body weight(1 mg/kg). Rats in the high-fat diet control group were injected withthe same volume of normal saline for injection in the same mannerdescribed above.

The injection sites mentioned above were the lower inguinal fat pads ofrats. Bilateral injections were administered evenly once a day on day 1,3, and 5 of the experiment. The rats were fed with high-fat diet for theentire duration of the experiment. The experiment lasted for 20 days,and the rats were euthanized on day 21 by CO2.

Please refer to FIG. 6. FIG. 6 is a bar graph showing the effects ofcurcumin-resveratrol complex subcutaneous injection formulationsprepared with different excipients on localized subcutaneous fat ofrats.

Results in FIG. 6 showed that the relative weight of the lower inguinalsubcutaneous fat of rats in the high-fat diet control group was100.0±13%, the relative weight of the lower inguinal subcutaneous fat ofrats in the low-dosage complex formula with formulation A group was134.9±39%, the relative weight of the lower inguinal subcutaneous fat ofrats in the high-dosage complex formula with formulation A group was134.9±14%, and the relative weight of the lower inguinal subcutaneousfat of rats in the low-dosage complex formula with ELP group was71.1±14%. Comparing to the high-fat diet control group, neither thelow-dosage complex formula with formulation A group nor high-dosagecomplex formula with formulation A group can reduce the fat at theadministration site (localized fat) (p>0.05).

The relative weight of the lower inguinal subcutaneous fat of rats inthe low-dosage complex formula with ELP group was significantlydifferent from that of rats in the control group (p<0.05), and therelative weight of the lower inguinal subcutaneous fat of rats in thelow-dosage complex formula with ELP group was reduced by 28.9%. Based onthe preparation described above, the curcumin-resveratrol complexsolution comprising ELP administered to the group comprises thedrug-containing micelles (encapsulating curcumin) and the secondlipophilic drug-containing micelles (encapsulating resveratrol).Therefore, similar to the curcumin simple pharmaceutical composition,formation of micelles is the critical factor for thecurcumin-resveratrol complex solution comprising ELP to significantlyreduce localized fat.

Experiment 7-3: Comparison Between Curcumin-Resveratrol ComplexPharmaceutical Composition and Simple Pharmaceutical Composition

The curcumin simple pharmaceutical composition, the resveratrol simplepharmaceutical composition, and the curcumin-resveratrol complexpharmaceutical composition of the present invention were prepared asfollows:

Preparation of the curcumin simple pharmaceutical composition: same asthe preparation of the curcumin ELP solution described in Experiment 2.Wherein, the concentration of curcumin was 5 mg/mL.

Preparation of the resveratrol simple pharmaceutical composition:approximately the same as the preparation of the curcumin ELP solutiondescribed in Experiment 2, only that curcumin was replaced byresveratrol. The concentration of resveratrol in the preparedresveratrol simple pharmaceutical composition was 5 mg/mL.

Preparation of the curcumin-resveratrol complex pharmaceuticalcomposition: same as the preparation of the curcumin-resveratrol complexsolution comprising ELP described in Experiment 7-2. Wherein, the totalconcentration of curcumin and resveratrol was 5 mg/mL, and the ratio ofcurcumin to resveratrol was 4:1.

The rats were assigned into a high-fat diet control group, a curcumingroup, a resveratrol group, and a curcumin-resveratrol complex group,with 5 rats in each group. The rats were fed in the same mannerdescribed in Experiment 2.

The curcumin simple pharmaceutical composition, the resveratrol simplepharmaceutical composition, and the curcumin-resveratrol complexpharmaceutical composition were injected to the lower inguinalsubcutaneous fat layer of rats in the curcumin group, the resveratrolgroup, and the curcumin-resveratrol complex group, respectively. Eachinjection volume was 2 mL per kilogram of body weight (2 mL/kg), suchthat each injected dosage was 10 mg of drug per kilogram of body weight(10 mg/kg). That is, rats in the curcumin group were administered with10 mg of curcumin per kilogram of body weight; rats in the resveratrolgroup was administered with 10 mg of resveratrol per kilogram of bodyweight; rats in the curcumin-resveratrol complex group were administeredwith 8 mg of curcumin and 2 mg of resveratrol per kilogram of bodyweight. Rats in the high-fat diet control group were injected with asame volume of normal saline for injection in the same manner describedabove.

The injection sites mentioned above were the lower inguinal fat pads ofrats. Bilateral injections were administered evenly once a day on day 1,2, 3, and 4 of the experiment. The rats were fed with high-fat diet forthe entire duration of the experiment. The experiment lasted for 14days, and the rats were euthanized on day 15 by CO2.

Because each group was administered with 10 mg/kg of drug each time, thelocal lipolysis efficacy of the curcumin-resveratrol complex groupshould be between that of the curcumin group and the resveratrol group.If the local lipolysis efficacy of the curcumin-resveratrol complexgroup is better than that of the curcumin group and the resveratrolgroup, it indicates that curcumin and resveratrol in thecurcumin-resveratrol complex pharmaceutical composition manifestssynergy in the local lipolysis efficacy.

Please refer to FIG. 7. FIG. 7 is a bar graph showing the effect ofcurcumin simple, resveratrol simple, and curcumin-resveratrol complexsubcutaneous injection formulations comprising micelles on the amount oflocalized subcutaneous fat of rats.

Results in FIG. 7 showed that the relative weight of the lower inguinalsubcutaneous fat in rats in the high-fat diet control group was100.0±14.6%, the relative weight of the lower inguinal subcutaneous fatof rats in the curcumin group was 93.5±6.5%, the relative weight of thelower inguinal subcutaneous fat of rats in the resveratrol group was91.6±27.8%, and the relative weight of the lower inguinal subcutaneousfat of rats in the curcumin-resveratrol complex group was 80.0±5.8%.Comparing to the high-fat diet control group, the curcumin or theresveratrol group cannot significantly reduce the fat at theadministration site (localized fat) (p>0.05).

The relative weight of the lower inguinal subcutaneous fat of rats inthe curcumin-resveratrol complex group was significantly different fromthat of rats in the high-fat diet control group (p<0.05), and therelative weight of the lower inguinal subcutaneous fat of rats in thecurcumin-resveratrol complex group was reduced by 20%.

Comparison among the local lipolysis efficacy of the curcumin group, theresveratrol group, and the curcumin-resveratrol complex groupdemonstrated that curcumin and resveratrol in the curcumin-resveratrolcomplex group manifests synergy in the local lipolysis efficacy.

Experiment 7-4: The Effects of Dosing Frequency on the Subcutaneous Fatand the Body Weight of Rats

In this experiment, rats in each group were administered with an equalamount of total injected dosage of the curcumin-resveratrol complexpharmaceutical composition but with different dosing frequency to assessthe effect of dosing frequency on the subcutaneous fat and the bodyweight of rats. In this experiment, other rats were administered withthe main ingredient of a local lipolysis injection formulation availablein the market to simultaneously compare the effects of thecurcumin-resveratrol complex pharmaceutical composition of the presentinvention and the local lipolysis injection formulation in the market onthe subcutaneous fat and the body weight of rats.

A sodium deoxycholate solution and a curcumin-resveratrol complexpharmaceutical composition were prepared as follows:

Preparation of the sodium deoxycholate solution: An appropriate amountof sodium deoxycholate was mixed with sterile water for injection tomake the concentration of sodium deoxycholate 2.575 mg/mL. The solutionwas mixed well to obtain the sodium deoxycholate solution. Wherein,sodium deoxycholate (purchased from Sigma-Aldrich, cat #D6750) is themain ingredient of the local lipolysis injection formulation ATX-101(brand name: Kybella) in the market.

Preparation of the curcumin-resveratrol complex pharmaceuticalcomposition: same as the preparation of the curcumin-resveratrol complexsolution comprising ELP described in Experiment 7-2. Wherein, the totalconcentration of curcumin and resveratrol was 5 mg/mL, and the ratio ofcurcumin to resveratrol was 4:1.

The rats were randomly assigned into 4 groups, which were a high-fatdiet control group, a sodium deoxycholate group, a high-dosing frequencycurcumin-resveratrol group (abbreviated as high-dosing frequency groupin this experiment), and a low-dosing frequency curcumin-resveratrolgroup (abbreviated as low-dosing frequency group in this experiment).The rats were fed in the same manner described in Experiment 2.

The drugs were administered as follows:

The sodium deoxycholate group: The sodium deoxycholate solution wasinjected to the lower inguinal subcutaneous fat layer of rats in thesodium deoxycholate group. Each injection volume was 4 mL per kilogramof body weight (4 mL/kg), such that each injected dosage was 10.3 mg(10.3 mg/kg; calculation: 2.575 mg/mL×4 mL/kg=10.3 mg/kg). Rats wereinjected once a day on day 1, 3, and 5 of the experiment, with 3injections in total, such that the total dosage was 30.9 mg/kg (10.3mg/kg×3 times=30.9 mg/kg).

The high-dosing frequency group: The curcumin-resveratrol complexpharmaceutical composition was injected to the lower inguinalsubcutaneous fat layer of rats in the high-dosing frequency group. Eachinjection volume was 4 mL per kilogram of body weight (4 mL/kg), suchthat each injected dosage was 20 mg (20 mg/kg; calculation: 5 mg/mL×4mL/kg=20 mg/kg). Rats were injected once a day on day 1, 3, 5, 7, 9, and11 of the experiment, with 6 injections in total, such that the totaldosage was 120 mg/kg (20 mg/kg×6 times=120 mg/kg).

The low-dosing frequency group: The curcumin-resveratrol complexpharmaceutical composition was injected to the lower inguinalsubcutaneous fat layer of rats in the low-dosing frequency group. Eachinjection volume was 8 mL per kilogram of body weight (8 mL/kg), suchthat each injected dosage was 40 mg (40 mg/kg; calculation: 5 mg/mL×8mL/kg=40 mg/kg). Rats were injected once a day on day 1, 3, and 5 of theexperiment, with 3 injections in total, such that the total dosage was120 mg/kg (40 mg/kg×3 times=120 mg/kg).

The high-fat diet control group: rats were injected with water forinjection in the same manner described above.

The rats were fed with high-fat diet for the entire duration of theexperiment. The experiment lasted for 20 days, and the rats wereeuthanized on day 21 by CO2.

Please refer to FIGS. 8A and 8B. FIG. 8A is a bar graph showing theeffects of dosing frequency of the curcumin-resveratrol complexpharmaceutical composition on localized subcutaneous fat of rats. FIG.8B is a bar graph showing the effects of dosing frequency of thecurcumin-resveratrol complex pharmaceutical composition on total bodyweight gain of rats.

Results in FIG. 8A showed that the relative weight of the lower inguinalsubcutaneous fat of rats in the high-dosing frequency group was100.0±22.6%, the relative weight of the lower inguinal subcutaneous fatof rats in the sodium deoxycholate group was 88.8±16.7%, the relativeweight of the lower inguinal subcutaneous fat of rats in the high-dosingfrequency group was 62.3±5.1%, and the relative weight of the loweringuinal subcutaneous fat of rats in the low-dosing frequency group was65.4±11.3%.

Comparing to the high-fat diet control group, both the high-dosingfrequency group and the low-dosing frequency group can significantlyreduce the fat at the administration site (localized fat) (p<0.05).Thus, if the concentration of the curcumin-resveratrol complexpharmaceutical composition is sufficient, low-dosing frequency canachieve the effect of local lipolysis.

Comparing to the low-dosing frequency group, the lipolysis effect of thehigh-dosing frequency group is better. Although there was no significantdifference between high-dosing frequency and low-dosing frequency, thehigh-dosing frequency can achieve a better trend of local lipolysiseffect.

Results in FIG. 8B showed that the relative weight gain in rats of thehigh-fat diet control group was 100.0±11.6%, the relative weight gain ofrats in the sodium deoxycholate group was 100.2±12.6%, the relativeweight gain of rats in the high-dosing frequency group was 63.5±5.5%,and the relative weight gain of rats in the low-dosing frequency groupwas 78.7±11.5%.

Comparing to the relative weight gain of rats in the high-fat dietcontrol group, the relative weight gain of rats in both the low-dosingfrequency group and the high-dosing frequency group was significantlydecreased (p<0.05), and the relative weight gain was decreased by 21.3%and 36.5%, respectively, showing that the weight loss effect was verysignificant.

Therefore, the curcumin-resveratrol complex pharmaceutical compositionof the present invention can significantly reduce the body weight, andthe weight loss efficacy of high-dosing frequency is significantlybetter than that of low-dosing frequency (p<0.05).

Based on the experiences of the inventor, when the dosing frequencysuitable for rats is 3-6 times, the dosing frequency suitable for humanis 1-12 times. Preferably, the dosing frequency for human is 1-6 times.

Preferably, the dosing frequency for human is 1-12 times every other dayto every 30 days. Preferably, the dosing frequency for human is 1-6times every other day to every 30 days. Or, preferably, the dosingfrequency for human is 3-60 times every other day to every 20 days;preferably, the dosing frequency for human is 6-42 times every other dayto every 14 days.

Experiment 7-5: The Effects of Administered Dosage on the SubcutaneousFat of Rats

In this experiment, rats were administered with different dosages of thecurcumin-resveratrol complex pharmaceutical composition to assess theeffects of administered dosage on the subcutaneous fat of rats.Additionally, in this experiment, other rats were administered with themain ingredient of another local lipolysis injection formulationcurrently undergoing clinical trials to simultaneously compare theeffects of the curcumin-resveratrol complex pharmaceutical compositionof the present invention and the other local lipolysis injectionformulation currently undergoing clinical trials on the subcutaneous fatof rats.

A ELP solution, a LIPO-202 solution, and the curcumin-resveratrolcomplex pharmaceutical composition were prepared as follows:

The ELP solution: 18 g of KOLLIPHOR® ELP (polyoxyl-35-castor oil,formerly known as CREMOPHOR® ELP, abbreviated as ELP) was mixed with anappropriate amount of normal saline for injection to a total volume of90 mL. The solution was mixed well to obtain the ELP solution. Wherein,the concentration of ELP was approximately 20%.

Preparation of the LIPO-202 solution:

-   -   LIPO-202 is a local lipolysis injection formulation currently        undergoing clinical trials, and its main ingredient is        salmeterol xinafonate.    -   (i) 1 mg of salmeterol xinafonate (purchased from Sigma-Aldrich)        was mixed with an appropriate amount of methanol to a total        volume of 1 mL to obtain a 1 mg/mL stock solution.    -   (ii) The stock solution was 10-fold serial diluted with sterile        water to prepare a salmeterol xinafonate solution of a final        concentration of 0.01 μg/mL, which is the LIPO-202 solution used        in this experiment.

Preparation of the curcumin-resveratrol complex pharmaceuticalcomposition: same as the preparation of the curcumin-resveratrol complexsolution comprising ELP described in Experiment 7-2. Wherein, the totalconcentration of curcumin and resveratrol was 5 mg/mL, and the ratio ofcurcumin to resveratrol was 4:1, and the concentration of ELP was 20%.

The rats were randomly assigned into 7 groups, which were a high-fatdiet control group, a control group, a LIPO-202 group, a 1 mg/mL complexformula group, a 5 mg/mL complex formula group, a 10 mg/mL complexformula group, and a 20 mg/mL complex formula group. The rats were fedin the same manner described in Experiment 2.

The drugs were administered as follows:

The control group: The ELP solution was injected to the lower inguinalsubcutaneous fat layer of rats in the control group. Each injectionvolume was 4 mL per kilogram of body weight (4 mL/kg).

The LIPO-202 group: The LIPO-202 solution was injected to the loweringuinal subcutaneous fat layer of rats in the LIPO-202 group. Eachinjection volume was 4 mL per kilogram of body weight (4 mL/kg), suchthat each injected dosage was 0.04 μg/kg (0.04 μg/kg; calculation: 0.01μg/mL×4 mL/kg=0.04 μg/kg).

The 1 mg/mL complex formula group: The curcumin-resveratrol complexpharmaceutical composition was injected to the lower inguinalsubcutaneous fat layer of rats in the 1 mg/mL complex formula group.Each injection volume was 0.2 mL per kilogram of body weight (0.2mL/kg), such that each injected dosage was 1 mg/kg (1 mg/kg;calculation: 5 mg/mL×0.2 mL/kg=1 mg/kg).

The 5 mg/mL complex formula group: The curcumin-resveratrol complexpharmaceutical composition was injected to the lower inguinalsubcutaneous fat layer of rats in the 5 mg/mL complex formula group.Each injection volume was 1 mL per kilogram of body weight (1 mL/kg),such that each injected dosage was 5 mg/kg (5 mg/kg; calculation: 5mg/mL×1 mL/kg=5 mg/kg).

The 10 mg/mL complex formula group: The curcumin-resveratrol complexpharmaceutical composition was injected to the lower inguinalsubcutaneous fat layer of rats in the 10 mg/mL complex formula group.Each injection volume was 2 mL per kilogram of body weight (2 mL/kg),such that each injected dosage was 10 mg/kg (10 mg/kg; calculation: 5mg/mL×2 mL/kg=10 mg/kg).

The 20 mg/mL complex formula group: The curcumin-resveratrol complexpharmaceutical composition was injected to the lower inguinalsubcutaneous fat layer of rats in the 20 mg/mL complex formula group.Each injection volume was 4 mL per kilogram of body weight (4 mL/kg),such that each injected dosage was 20 mg/kg (20 mg/kg; calculation: 5mg/mL×4 mL/kg=20 mg/kg).

The high-fat diet control group: rats were injected with water forinjection in the same manner described above.

Rats were injected once a day on day 1, 2, 3, and 4 of the experiment.The rats were fed with high-fat diet for the entire duration of theexperiment. The experiment lasted for 14 days, and the rats wereeuthanized on day 15 by CO2.

Please refer to FIG. 9. FIG. 9 is a bar graph showing the effects ofadministered dosage of the curcumin-resveratrol complex pharmaceuticalcomposition on localized subcutaneous fat of rats.

Results in FIG. 9 showed that the relative weight of the lower inguinalsubcutaneous fat of rats in the high-fat diet control group was100±15.2%, the relative weight of the lower inguinal subcutaneous fat ofrats in the control group was 99.2±22.0%, the relative weight of thelower inguinal subcutaneous fat of rats in the LIPO-202 group was97.8±12.8%, the relative weight of the lower inguinal subcutaneous fatof rats in the 1 mg/mL complex formula group was 90.1±12.2%, therelative weight of the lower inguinal subcutaneous fat of rats in the 5mg/mL complex formula group was 80.9±13.9%, the relative weight of thelower inguinal subcutaneous fat of rats in the 10 mg/mL complex formulagroup was 73.9±9.5%, and the relative weight of the lower inguinalsubcutaneous fat of rats in the 20 mg/mL complex formula group was64.1±12.0%.

Therefore, the curcumin-resveratrol complex pharmaceutical compositionachieved a significant local lipolysis effect at a dosage of 5 mg/kg,and the effects were more significant depend on the higher dosage.Although the dosage of 1 mg/kg of curcumin-resveratrol complexpharmaceutical composition did not achieve significant local lipolysiseffect, but it induced a trend. Based on the experiences of theinventor, if dosing frequency is increased, the curcumin-resveratrolcomplex pharmaceutical composition can also achieve significant locallipolysis effect at a dosage of 1 mg/kg.

Base on the experiences of the inventor, when the administered dosagesuitable for rats is 1 mg/kg-20 mg/kg, the administered dosage suitablefor human is 0.01-40 mg/kg. Preferably, the administered dosage suitablefor human is 0.1-20 mg/kg.

Preferably, the administered dosage suitable for human is 0.02-20mg/cm2. Preferably, the administered dosage suitable for human is0.04-16 mg/cm2. Preferably, the administered dosage suitable for humanis 0.2-12 mg/cm2. Preferably, the administered dosage suitable for humanis 0.4-8 mg/cm2.

Preferably, the administered dosage suitable for human is 0.01-40 mg/kgof body weight. Preferably, the administered dosage suitable for humanis 0.4-40 mg/kg of body weight. Preferably, the administered dosagesuitable for human is 0.8-20 mg/kg of body weight.

Experiment 8: The Effects of Curcumin Complex PharmaceuticalCompositions on Lipolysis

This experiment used curcumin and lipophilic drugs other thanresveratrol to prepare complex pharmaceutical compositions to assess thelipolysis efficacy of various lipophilic complex pharmaceuticalcompositions on mature adipocytes.

This experiment chose to use puerarin, quercetin, and synephrine toprepare various lipophilic complex pharmaceutical compositions.

Experiment 8-1 Cytotoxicity Test

-   -   Determine if 50 ppm of curcumin, puerarin, quercetin, or        synephrine have toxicity to cells other than adipocytes by MTT        assay. Only if the drug is deemed non-toxic will lipolysis test        be proceeded.

Experimental results showed that 50 ppm of curcumin, puerarin,quercetin, and synephrine are not cytotoxic to rat somatic cells otherthan adipocytes, so this dosage will not affect the general somaticcells.

Experiment 8-2 Lipolysis Efficacy on Mature Adipocytes

A DMSO control group cell culture medium, a curcumin cell culturemedium, a puerarin cell culture medium, a quercetin cell culture medium,a synephrine cell culture medium, a curcumin-puerarin complex cellculture medium, a curcumin-quercetin complex cell culture medium, and acurcumin-synephrine complex cell culture medium were prepared asfollows:

The DMSO control group cell culture medium: DMSO was mixed with anappropriate amount of sterile water to obtain a 0.5% DMSO solution. The0.5% DMSO solution was mixed with a cell culture medium (product name:Dulbecco's Modified Eagle Medium, purchased from Gibco) to prepare theDMSO control group cell culture medium, wherein, the volume ratiobetween the 0.5% DMSO solution and the cell culture medium was 1:1000.

The curcumin cell culture medium: curcumin was mixed with an appropriateamount of 0.5% DMSO solution to obtain a curcumin solution. The curcuminsolution was mixed with a cell culture medium (product name: Dulbecco'sModified Eagle Medium, purchased from Gibco) to prepare the curcumincell culture medium containing 50 ppm of curcumin, wherein, the volumeratio between the curcumin solution and the cell culture medium was1:1000.

The puerarin cell culture medium: puerarin (purchased fromSigma-Aldrich) was mixed with an appropriate amount of 0.5% DMSOsolution to obtain a puerarin solution. The puerarin solution was mixedwith a cell culture medium to prepare the puerarin cell culture mediumcontaining 50 ppm of puerarin, wherein, the volume ratio between thepuerarin solution and the cell culture medium is 1:1000.

The quercetin cell culture medium: quercetin (purchased fromSigma-Aldrich) was mixed with an appropriate amount of 0.5% DMSOsolution to obtain a quercetin solution. The quercetin solution wasmixed with a cell culture medium to prepare the quercetin cell culturemedium containing 50 ppm of quercetin, wherein, the volume ratio betweenthe quercetin solution and the cell culture medium was 1:1000.

The synephrine cell culture medium: synephrine (purchased fromSigma-Aldrich) was mixed with an appropriate amount of 0.5% DMSOsolution to obtain a synephrine solution. The synephrine solution wasmixed with a cell culture medium to prepare the synephrine cell culturemedium containing 50 ppm of synephrine, wherein, the volume ratiobetween the synephrine solution and the cell culture medium was 1:1000.

The curcumin-puerarin complex cell culture medium: curcumin and puerarinwere mixed with an appropriate amount of 0.5% DMSO solution to obtain acurcumin-puerarin complex solution. Wherein, the weight ratio betweencurcumin and puerarin was 2:3. The curcumin-puerarin complex solutionwas mixed with a cell culture medium to prepare the curcumin-puerarincomplex cell culture medium containing 50 ppm of curcumin-puerarincomplex drug, wherein, the concentration of curcumin was 20 ppm, theconcentration of puerarin was 30 ppm, and the volume ratio between thecurcumin-puerarin complex solution and the cell culture medium was1:1000.

The curcumin-quercetin complex cell culture medium: curcumin andquercetin were mixed with an appropriate amount of 0.5% DMSO solution toobtain a curcumin-quercetin complex solution. Wherein, the weight ratiobetween curcumin and quercetin was 2:3. The curcumin-quercetin complexsolution was mixed with a cell culture medium to prepare thecurcumin-quercetin complex cell culture medium containing 50 ppm ofcurcumin-quercetin complex drug, wherein, the concentration of curcuminwas 20 ppm, the concentration of quercetin was 30 ppm, and the volumeratio between the curcumin-quercetin complex solution and the cellculture medium was 1:1000.

The curcumin-synephrine complex cell culture medium: curcumin andsynephrine were mixed with an appropriate amount of 0.5% DMSO solutionto obtain a curcumin-synephrine complex solution. Wherein, the weightratio between curcumin and synephrine was 2:3. The curcumin-synephrinecomplex solution was mixed with a cell culture medium to prepare thecurcumin-synephrine complex cell culture medium containing 50 ppm ofcurcumin-synephrine complex drug, wherein, the concentration of curcuminwas 20 ppm, the concentration of synephrine was 30 ppm, and the volumeratio between the curcumin-synephrine complex solution and the cellculture medium was 1:1000.

Experimental Procedure to Determine the Lipolysis Efficacy on MatureAdipocytes

-   -   The adipocyte precursors 3T3-L1 cells (purchased from the Food        Industry Research and Development Institute. Taiwan; abbreviated        as BCRC) were seeded in 12-well plates, such that each well        contained 1×105 cells. After two days of culture, the cells were        cultured for another two days in a cell differentiation        induction media (DMI medium, wherein contains 0.5 pM of IBMX        (purchased from Sigma-Aldrich), 0.1 pM of dexamethasone        (purchased from Sigma-Aldrich), and 5 μg/ml of insulin        (purchased from Humulin R.)) Then, the cells were cultured in a        medium containing 5 μg/ml of insulin. Once the cell morphology        changed from spindle-shaped to spherical and many lipid droplets        were accumulated in the cells, it indicated that the cells have        differentiated into mature adipocytes.

The mature adipocytes were assigned into 8 groups, which were a DMSOcontrol group, a curcumin group, a puerarin group, a quercetin group, asynephrine group, a curcumin-puerarin complex group, acurcumin-quercetin complex group, and a curcumin-synephrine complexgroup.

The mature adipocytes in the DMSO control group, the curcumin group, thepuerarin group, the quercetin group, the synephrine group, thecurcumin-puerarin complex group, the curcumin-quercetin complex group,and the curcumin-synephrine complex group were respectively culturedwith the DMSO control group cell culture medium, the curcumin cellculture medium, the puerarin cell culture medium, the quercetin cellculture medium, the synephrine cell culture medium, thecurcumin-puerarin complex cell culture medium, the curcumin-quercetincomplex cell culture medium, and the curcumin-synephrine complex cellculture medium for 24 hours.

Annexin V protein (purchased from eBioscience) and propidium iodide (PI;purchased from eBioscience) were mixed with the cells in each group fora period of time, and then the percentage of cells labeled by annexin Vprotein and PI in each group was analyzed by flow cytometry to assessthe percentage of mature adipocytes undergoing apoptosis. Wherein, whena mature adipocyte is labeled by both annexin V protein and PI, itindicates that the cell is undergoing apoptosis; when more matureadipocytes are undergoing apoptosis, it indicates that the lipolysisefficacy of the administered drug is better, and it also indicates thatlipolysis is mediated through apoptosis but not necrosis.

The data were presented as mean f SD and analyzed by one-way ANOVA.Statistical results were shown as symbols or letters. Different symbolsor letters indicates statistically significant difference (p<0.05), andidentical symbols or letters indicates no statistically significantdifference (p>0.05).

Because the total dosage of the administered drug in each group was 50ppm, the apoptosis efficacy of the curcumin-puerarin complex groupshould be between the efficacy of the curcumin group and the pueraringroup. If the apoptosis efficacy of the curcumin-puerarin complex groupis better than that of the curcumin group and the puerarin group, itindicates that curcumin and puerarin in the curcumin-puerarin complexgroup manifests synergy in lipolysis efficacy. Similarly, the apoptosisefficacy of the curcumin-quercetin complex group should be between theefficacy of the curcumin group and the quercetin group. If the apoptosisefficacy of the curcumin-quercetin complex group is better than that ofthe curcumin group and the quercetin group, it indicates that curcuminand quercetin in the curcumin-quercetin complex group manifests synergyin lipolysis efficacy. The apoptosis efficacy of the curcumin-synephrinecomplex group should be between the efficacy of the curcumin group andthe synephrine group. If the apoptosis efficacy of thecurcumin-synephrine complex group is better than that of the curcumingroup and the synephrine group, it indicates that curcumin andsynephrine in the curcumin-synephrine complex group manifests synergy inlipolysis efficacy.

Please refer to FIG. 10. FIG. 10 is a bar graph showing the effects ofcurcumin-other lipophilic drug complex pharmaceutical compositions onpromoting apoptosis of mature adipocytes

Results in FIG. 10 showed that the percentage of apoptotic cells of theDMSO control group was 0.8±0.2%, the percentage of apoptotic cells ofthe curcumin group was 78.4±5.4%, the percentage of apoptotic cells ofthe puerarin group was 2.0±1.6%, the percentage of apoptotic cells ofthe quercetin group was 1.8±0.6%, the percentage of apoptotic cells inthe synephrine group was 0.9±0.2%, the percentage of apoptotic cells ofthe curcumin-puerarin complex group was 80.0±5.9%, the percentage ofapoptotic cells of the curcumin-quercetin complex group was 80.4±7.0%,and the percentage of apoptotic cells of the curcumin-synephrine complexgroup was 80.8±4.8%.

Comparison among the apoptosis efficacy of the curcumin group, thepuerarin group, and the curcumin-puerarin complex group demonstratedthat curcumin and puerarin in the curcumin-puerarin complexpharmaceutical composition manifests synergy in lipolysis efficacy.

Comparison among the apoptosis efficacy of the curcumin group, thequercetin group, and the curcumin-quercetin complex group demonstratedthat curcumin and quercetin in the curcumin-quercetin complexpharmaceutical composition manifests synergy in lipolysis efficacy.

Comparison among the apoptosis efficacy of the curcumin group, thesynephrine group, and the curcumin-synephrine complex group demonstratedthat curcumin and synephrine in the curcumin-quercetin complexpharmaceutical composition manifests synergy in lipolysis efficacy.

Therefore, complex pharmaceutical compositions formed by curcumin andvarious lipophilic drugs can all achieve the effect of lipolysis, andthere are synergies between curcumin and various lipophilic drugs in thelipolysis efficacy. Therefore, the present invention uses curcumin andvarious lipophilic drugs to prepare drug-containing micelles and asecond lipophilic drug-containing micelles, and further prepares thecurcumin-other lipophilic drug complex pharmaceutical compositions,which are the pharmaceutical compositions capable of being used forlocalized lipolysis and weight reduction.

The present invention provides a first preparation method for preparinga curcumin-other lipophilic drug complex pharmaceutical composition, andthe curcumin-other lipophilic drug complex pharmaceutical compositioncomprises drug-containing micelles and the second lipophilicdrug-containing micelles; the procedure of the first preparation toprepare the curcumin-other lipophilic drug complex pharmaceuticalcomposition is as follows:

-   -   (A) Steps to prepare drug-containing micellar subassembly, to        prepare a drug-containing micellar subassembly;    -   (B) Steps to prepare a second lipophilic drug-containing        micellar subassembly, to prepare the second lipophilic        drug-containing micellar subassembly; and    -   (C) Mixing the drug-containing micellar subassembly with the        second lipophilic drug-containing micellar subassembly, to        prepare the curcumin-other lipophilic drug complex        pharmaceutical composition;

Wherein, the step (A) to prepare the drug-containing micellarsubassembly comprises the following steps (a2)-(d2)

-   -   (a2) Mixing curcumin with a first solvent, and stirring at        150-500 rpm at room temperature until curcumin dissolves        completely;    -   (b2) Adding a first pharmaceutically acceptable surfactant, and        stirring well at 100 300 rpm to volatilize the first solvent,        wherein, the hydrophilic-lipophilic balance value (HLB value) of        the first surfactant is greater than 10;    -   (c2) After the first solvent volatilizes completely, obtaining        the drug-containing micelles; and    -   (d2) Filtering through a 0.2 um filter, and the filtered        solution is the drug-containing micellar subassembly comprising        drug-containing micelles:

Moreover, the step (B) to prepare the second lipophilic drug-containingmicellar subassembly comprises the following steps (a3)-(d3):

-   -   (a3) Mixing a second lipophilic drug with a second solvent, and        stirring at 200 500 rpm at room temperature until the second        lipophilic drug dissolves completely;    -   (b3) Adding a second pharmaceutically acceptable surfactant, and        stirring well at 100-300 rpm to volatilize the second solvent,        wherein the hydrophilic-lipophilic balance value (HLB value) of        the second surfactant is greater than 10;    -   (c3) After the second solvent volatilizes completely, obtaining        the second lipophilic drug-containing micelles; and    -   (d3) Filtering through a 0.2 um filter, and the filtered        solution is the second lipophilic drug-containing micellar        subassembly comprising the second lipophilic drug-containing        micelles

Wherein, in step (c2), the drug-containing micelle is a microstructureformed by a surfactant, and curcumin is encapsulated in saiddrug-containing micelle. In step (c3), the second lipophilicdrug-containing micelle is a microstructure formed by the secondsurfactant, and the second lipophilic drug is encapsulated in saidsecond lipophilic drug-containing micelle.

Preferably, the operating procedure of step (c2) is: After the firstsolvent volatilizes completely, slowly adding a pharmaceuticallyacceptable aqueous solution and mixing well to form drug-containingmicelles.

Preferably, the operating procedure of step (c3) is: After the secondsolvent volatilizes completely, slowly adding a pharmaceuticallyacceptable aqueous solution and mixing well to form the secondlipophilic drug-containing micelles.

Preferably, the second lipophilic drug is at least one of quercetin,synephrin, puerarin, resveratrol, and any lipophilic drug exceptcurcumin, or combination thereof.

Preferably, in step (a2) and/or step (a3), the boiling point(s) of thefirst solvent and/or the second solvent are/is lower than the boilingpoint of pure water.

Preferably, in step (a2) and/or step (a3), the first solvent and/or thesecond solvent are/is a hydrophilic solvent.

Preferably, the hydrophilic solvent is at least one of methanol,ethanol, acetone, and other hydrophilic solvents, or combinationthereof.

Preferably, in step (a2) and/or step (a3), the first solvent and/or thesecond solvent are/is a lipophilic solvent.

Preferably, the lipophilic (hydrophobic) solvent is at least one ofether, benzene, chloroform, dichloromethane, hexane, and otherlipophilic (hydrophobic) solvents, or combination thereof.

Preferably, in step (b2) and/or (b3), the first surfactant and/or thesecond surfactant are/is a non-ionic surfactant.

Preferably, the non-ionic surfactant is at least one of polysorbate 80(TWEEN® 80), polyoxyl 15 hydroxystearate (KOLLIPHOR® HS 15),polyoxyethylene castor oil derivatives, and other non-ionic surfactants,or combination thereof.

Preferably, the polyoxyethylene castor oil derivative is at least one ofKOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP), KOLLIPHOR® RH 40 (polyoxyl 40 hydrogenated castor oil), and otherpolyoxyethylene castor oil derivatives, or combination thereof.

Preferably, the weight ratio of curcumin to the second lipophilic drugis 30:1 1:10.

Preferably, in steps (a2) and (b2), the weight ratio of the curcumin andthe first surfactant is 1:4 to 1:500.

Preferably, in steps (a3) and (b3), the weight ratio of the secondlipophilic drug and the second surfactant is 1:4 to 1:500.

Preferably, in step (c2) and/or (c3), the pharmaceutically acceptableaqueous solution is water for injection, aqueous solution for injection,or normal saline.

Preferably, in step (c2) and/or (c3), the pharmaceutically acceptableaqueous solution comprises a local anesthetic.

Preferably, the local anesthetic is at least one of amides,para-aminobenzoic acid esters, and amino ethers, or combination thereof

Preferably, the amides are at least one of dibucaine, lidocaine,mepivacaine HCl, bupivacine HCl, pyrrocaine HCl, Prilocaine HCl,digammacaine, and oxethazaine, or combination thereof.

Preferably, the para-aminobenzoic acid esters are at least one ofbutacaine, dimethocaine, and tutocaine, or combination thereof.

Preferably, the amino ethers are at least one of quinisocaine andpramocaine, or combination thereof.

Preferably, in step (c2) and/or (c3), the pharmaceutically acceptableaqueous solution comprises an antioxidant.

Preferably, the antioxidant is at least one of beta-carotene, lutein,lycopene, bilirubin, vitamin A, vitamin C (ascorbic acid), vitamin E,uric acid, nitric oxide, nitroxide, pyruvate, catalase, superoxidedismutase, glutathione peroxidases, N-acetyl cysteine, and naringenin,or combination thereof.

The present invention provides a second preparation of thecurcumin-other lipophilic drug complex pharmaceutical composition, andthe second preparation method of the curcumin-other lipophilic drugcomplex pharmaceutical composition is more concise than the firstpreparation method of the curcumin-other lipophilic drug complexpharmaceutical composition: the procedure of the second preparationmethod for the curcumin-other lipophilic drug complex pharmaceuticalcomposition is as follows:

-   -   (a4) Mixing curcumin, the second lipophilic drug, and a solvent,        and stirring at 200-500 rpm until curcumin dissolves completely;    -   (b4) Adding a pharmaceutically acceptable surfactant and        stirring well at 100-300 rpm to volatilize the solvent, wherein,        the hydrophilic-lipophilic balance value (HLB value) of the        surfactant is greater than 10;    -   (c4) Once the solvent volatilizes completely, slowly adding a        pharmaceutically acceptable aqueous solution and mixing well to        form drug-containing micelles and the second lipophilic        drug-containing micelles; and    -   (d4) Filtering through a 0.2 um filter, and storing the filtered        solution comprising the drug-containing micelles and the second        lipophilic drug-containing micelles in dark and refrigeration.

The types and ranges of the solvents, the surfactants, thepharmaceutically acceptable aqueous solutions, and the second lipophilicdrugs used in the second preparation for the curcumin-other lipophilicdrug complex pharmaceutical composition are the same as those used inthe first preparation of the curcumin-other lipophilic drug complexpharmaceutical composition. Additionally, the ranges of relative ratiosof the ingredients used in the second preparation of the curcumin-otherlipophilic drug complex pharmaceutical composition are the same as thoseof the first preparation of the curcumin-other lipophilic drug complexpharmaceutical composition.

Preferably, the pharmaceutical composition comprises a local aestheticand/or an antioxidant.

Preferably, the types and ranges of the local anesthetics and theantioxidants of the second preparation of the curcumin-other lipophilicdrug complex pharmaceutical composition are the same as those used inthe first preparation of the curcumin-other lipophilic drug complexpharmaceutical composition.

Experiment 9: The Effects of Curcumin-Green Tea Extract ComplexSubcutaneous Injection Formulation on the Subcutaneous Fat of RatsExperiment 9-1: The Effects of Green Tea Extract Simple SubcutaneousInjection Formulation on the Subcutaneous Fat of Rats

Preparation of the Green Tea Extract Subcutaneous Injection Formulation:

Green tea extract was mixed with an appropriate amount of normal salinefor injection to obtain the green tea extract subcutaneous injectionformulation.

The rats were assigned into a high-fat diet control group and a greentea extract group with 6 rats in each group. The rats were fed in thesame manner described in Experiment 2. The green tea extractsubcutaneous injection formulation was injected into the lower inguinalsubcutaneous fat layer of rats in the green tea extract group. Eachinjected dosage was 8 mg of green tea extract per kilogram of bodyweight (8 mg/kg). Rats in the high-fat diet control group were injectedwith the same volume of water for injection in the same manner describedabove.

The injection sites mentioned above were the lower inguinal fat pads ofrats. Bilateral injections were administered evenly once a day on day 1,3, and 5 of the experiment. The rats were fed with high-fat diet for theentire duration of the experiment. Their weight changes were recordeddaily, and food and water consumption was recorded weekly. Theexperiment lasted for 20 days, and the rats were euthanized on day 21 byCO2.

Please refer to FIG. 11. FIG. 11 is a bar graph showing the effects ofthe green tea extract subcutaneous injection formulation withoutexcipient on localized subcutaneous fat of rats.

Results in FIG. 11 showed that the relative weight of the lower inguinalsubcutaneous fat of rats in the high-fat diet control group was100.00±21.51%, the relative weight of the lower inguinal subcutaneousfat of rats in the green tea extract group was 99.50±13.14%. There wasno significant difference between the relative weight of the loweringuinal subcutaneous fat of rats in the green tea extract group andthat of rats in the high-fat diet control group, indicating that ahydrophilic plant extract-green tea extract composition withoutexcipient cannot reduce the fat at the administration site (localizedfat).

Experiment 9-2: The Effects of Dosing Frequency on the Subcutaneous Fatand the Body Weight of Rats

In this experiment, rats in each group were administered with an equalamount of total injected dosage of the curcumin-green tea extractcomplex pharmaceutical composition but with different dosing frequencyto assess the effects of dosing frequency on the subcutaneous fat andthe body weight of rats. In this experiment, other rats wereadministered with the main ingredient of a local lipolysis injectionformulation available in the market to simultaneously compare theeffects of the curcumin-green tea extract complex pharmaceuticalcomposition of the present invention and the local lipolysis injectionformulation in the market on the subcutaneous fat and the body weight ofrats.

A sodium deoxycholate solution and the curcumin-green tea extractcomplex pharmaceutical composition were prepared as follows:

Preparation of the Sodium Deoxycholate Solution: Same as the Preparationof the Sodium Deoxycholate Solution in Experiment 7-4.

Preparation of the curcumin-green tea extract complex pharmaceuticalcomposition: 0.8 g of curcumin was mixed with 150-200 mL ofdichloromethane and stirred at 150-500 rpm to completely dissolvecurcumin. 40 g of KOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly knownas CREMOPHOR® ELP, abbreviated as ELP) was added and stirred at 100-300rpm to volatilize dichloromethane. Once dichloromethane volatilizedcompletely, normal saline for injection was slowly added to a totalvolume of 200 mL. Wherein, the normal saline for injection comprised 0.2g of green tea extract. The solution was mixed well to obtain thecurcumin-green tea extract complex solution comprising ELP. Saidcurcumin-green tea extract complex solution comprising ELP compriseddrug-containing micelles, the total concentration of curcumin and greentea was 5 mg/mL, and the weight ratio of curcumin and green tea extractwas 4:1

The rats were randomly assigned into 4 groups, which were a high-fatdiet control group, a sodium deoxycholate group, a high-dosing frequencycurcumin-green tea extract group (abbreviated as high-dosing frequencygroup in this experiment), and a low-dosing frequency curcumin-green teaextract group (abbreviated as low-dosing frequency group). The rats werefed in the same manner described in Experiment 2.

The drugs were administered as follows:

The sodium deoxycholate group: The sodium deoxycholate solution wasinjected to the lower inguinal subcutaneous fat layer of rats in thesodium deoxycholate group. Each injection volume was 4 mL per kilogramof body weight (4 mL/kg), such that each injected dosage was 10.3 mg/kg(10.3 mg/kg; calculation: 2.575 mg/mL×4 mL/kg=10.3 mg/kg). Rats wereinjected once a day on day 1, 3, and 5 of the experiment, with 3injections in total, such that the total dosage was 30.9 mg/kg (10.3mg/kg×3 times=30.9 mg/kg).

The high-dosing frequency group: The curcumin-green tea extract complexpharmaceutical composition was injected to the lower inguinalsubcutaneous fat layer of rats in the high-dosing frequency group. Eachinjection volume was 4 mL per kilogram of body weight (4 mL/kg), suchthat each injected dosage was 20 mg/kg (20 mg/kg; calculation: 5 mg/mL×4mL/kg=20 mg/kg). Rats were injected once a day on day 1, 3, 5, 7, 9, and11 of the experiment, with 6 injections in total, such that the totaldosage was 120 mg/kg (20 mg/kg×6 times=120 mg/kg).

The low-dosing frequency group: The curcumin-green tea extract complexpharmaceutical composition was injected to the lower inguinalsubcutaneous fat layer of rats in the low-dosing frequency group. Eachinjection volume was 8 mL per kilogram of body weight (8 mL/kg), suchthat each injected dosage was 40 mg/kg (40 mg/kg; calculation: 5 mg/mL×8mL/kg=40 mg/kg).

Rats were injected once a day on day 1, 3, and 5 of the experiment, with3 injections in total, such that the total dosage was 120 mg/kg (40mg/kg×3 times=120 mg/kg).

The high-fat diet control group: rats were injected with water forinjection in the same manner described above.

The rats were fed with high-fat diet for the entire duration of theexperiment. The experiment lasted for 20 days, and the rats wereeuthanized on day 21 by CO2.

Please refer to FIGS. 12A and 12B. FIG. 12A is a bar graph showing theeffects of the dosing frequency of the curcumin-green tea extractcomplex pharmaceutical composition on localized subcutaneous fat ofrats. FIG. 12B is a bar graph showing the effect of the dosing frequencyof the curcumin-green tea extract complex pharmaceutical composition ontotal body weight gain of rats.

Results in FIG. 12A showed that the relative weight of the loweringuinal subcutaneous fat of rats in the high-fat diet control group was100.0±22.6%, the relative weight of the lower inguinal subcutaneous fatof rats in the sodium deoxycholate group was 88.8±16.7%, the relativeweight of the lower inguinal subcutaneous fat of rats in the high-dosingfrequency group was 57.6±7.4%, and the relative weight of the loweringuinal subcutaneous fat of rats in the low-dosing frequency group was60.7±4.0%.

Comparing to the high-fat diet control group, both the high-dosingfrequency group and the low-dosing frequency group can significantlyreduce the fat at the administration site (localized fat) (p<0.05).Thus, if the concentration of the curcumin-green tea extract complexpharmaceutical composition is sufficient, low-dosing frequency canachieve the effect of local lipolysis.

Comparing to the low-dosing frequency group, the lipolysis effect of thehigh-dosing frequency group is better. Although there was no significantdifference between high-dosing frequency and low-dosing frequency, thehigh-dosing frequency can achieve a better trend of local lipolysiseffect.

Results in FIG. 12B showed that the relative weight gain of rats in thehigh-fat diet control group was 100.0±11.6%, the relative weight gain ofrats in the sodium deoxycholate group was 100.2±12.6%, the relativeweight gain of rats in the high-dosing frequency group was 58.7±9.0%,and the relative weight gain of rats in the low-dosing frequency groupwas 74.9±9.0%. Comparing to the relative weight gain of rats in thehigh-fat diet control group, the relative weight gain of rats in boththe low-dosing frequency group and the high-dosing frequency group wassignificantly decreased (p<0.05), and the relative weight gain wasreduced by 25.1% and 41.3%, respectively, showing that the weight losseffect was very significant.

Therefore, the curcumin-green tea extract complex pharmaceuticalcomposition of the present invention can significantly reduce the bodyweight, and the weight loss efficacy of high-dosing frequency issignificantly better than that of low-dosing frequency (p<0.05).

Based on the experiences of the inventor, when the dosing frequencysuitable for rats is 3-6 times, the dosing frequency suitable for humanis 1-12 times. Preferably, the dosing frequency for human is 1-6 times.

Preferably, the dosing frequency for human is 1-12 times every other dayto every 30 days. Preferably, the dosing frequency for human is 1-6times every other day to every 30 days. Or, preferably, the dosingfrequency for human is 3-60 times every other day to every 20 days;preferably, the dosing frequency for human is 6-42 times every other dayto every 14 days.

Experiment 10: The Effects of Curcumin Complex PharmaceuticalCompositions on Lipolysis

Curcumin and hydrophilic drugs expect green tea extract were used inthis experiment to prepare complex pharmaceutical compositions to assessthe lipolysis efficacy of various hydrophilic complex pharmaceuticalcompositions on mature adipocytes.

This experiment uses caffeine and L-carnitine to prepare varioushydrophilic complex pharmaceutical compositions.

Experiment 10-1: Cytotoxicity Test

-   -   Determine if 50 ppm of caffeine and L-carnitine have toxicity to        cells other than adipocytes by MTT assay. Only if the drug is        deemed non-toxic will lipolysis test be proceeded.

Experimental results showed that 50 ppm of caffeine and L-carnitine arenot cytotoxic to rat somatic cells expect adipocytes, so this dosagewill not affect the general somatic cells.

Experiment 10-2: Lipolysis Efficacy on Mature Adipocytes

A sterile water control group cell culture medium, a curcumin cellculture medium, a caffeine cell culture medium, an L-carnitine cellculture medium, a curcumin-caffeine complex cell culture medium, and acurcumin-L-carnitine complex cell culture medium were prepared asfollows:

The sterile water control group cell culture medium: Sterile water wasmixed with a cell culture medium to prepare the sterile water controlgroup cell culture medium. Wherein, the volume ratio of sterile waterand the cell culture medium was 1:1000.

The curcumin cell culture medium: same as the preparation of thecurcumin cell culture medium in Experiment 8-2.

The caffeine cell culture medium: Caffeine (purchased fromSigma-Aldrich) was mixed with an appropriate amount of sterile water toobtain a caffeine solution. The caffeine solution was mixed with a cellculture medium to prepare the caffeine cell culture medium containing 50ppm of caffeine. Wherein, the volume ratio of the caffeine cell culturemedium and the cell culture medium was 1:1000.

The L-carnitine cell culture medium: L-carnitine (purchased fromSigma-Aldrich) was mixed with an appropriate amount of sterile water toobtain a L-carnitine solution. The L-carnitine solution was mixed with acell culture medium to prepare the caffeine cell culture mediumcontaining 50 ppm of L-carnitine. Wherein, the volume ratio of theL-carnitine cell culture medium and the cell culture medium was 1:1000.

The curcumin-caffeine complex cell culture medium: Curcumin and caffeinewere mixed with an appropriate amount of sterile water to prepare acurcumin-caffeine complex solution. Wherein, the weight ratio ofcurcumin and caffeine was 2:3. The curcumin-caffeine complex solutionwas mixed with a cell culture medium to prepare the curcumin-caffeinecomplex cell culture medium containing 50 ppm of curcumin-caffeinecomplex drug. Wherein, the concentration of curcumin was 20 ppm, theconcentration of caffeine was 30 ppm, and the volume ratio of thecurcumin-caffeine complex solution and the cell culture medium was1:1000.

The curcumin-L-carnitine complex cell culture medium: Curcumin andL-carnitine were mixed with an appropriate amount of sterile water toprepare a curcumin-L-carnitine complex solution. Wherein, the weightratio of curcumin and L-carnitine was 2:3. The curcumin-L-carnitinecomplex solution was mixed with a cell culture medium to prepare thecurcumin-L-carnitine complex cell culture medium containing 50 ppm ofcurcumin-L-carnitine complex drug. Wherein, the concentration ofcurcumin was 20 ppm, the concentration of L-carnitine was 30 ppm, andthe volume ratio of the curcumin-L-carnitine complex solution and thecell culture medium was 1:1000.

Preparation of the Mature Adipocytes was the Same as that of Experiment8-2.

The adipocytes were assigned into 6 groups, which were a sterile watergroup, a curcumin group, a caffeine group, an L-carnitine group, acurcumin-caffeine complex group, and a curcumin-L-carnitine complexgroup.

The mature adipocytes in the sterile water group, the curcumin group,the caffeine group, the L-carnitine group, the curcumin-caffeine complexgroup, and the curcumin-L-carnitine complex group were cultured with thesterile water control group cell culture medium, the curcumin cellculture medium, the caffeine cell culture medium, the L-carnitine cellculture medium, the curcumin-caffeine complex cell culture medium, andthe curcumin-L-carnitine complex cell culture medium, respectively, for24 hours.

Annexin V protein (purchased from eBioscience) and propidium iodide (PI;purchased from eBioscience) were mixed with the cells in each group fora period of time, and then the percentage of cells labeled by annexin Vprotein and PI in each group was analyzed by flow cytometry to assessthe percentage of mature adipocytes undergoing apoptosis. Wherein, whena mature adipocyte is labeled by both annexin V protein and PI, itindicates that the cell is undergoing apoptosis; when more matureadipocytes are undergoing apoptosis, it indicates that the lipolysisefficacy of the administered drug is better, and it also indicates thatlipolysis is mediated through apoptosis but not necrosis.

Because the total dosage of the administered drug was 50 ppm, and 40%was curcumin and 60% was caffeine, therefore, the lipolysis efficacy ofthe curcumin-caffeine complex group should be close to the average ofthat of the curcumin group and the caffeine group. If the lipolysisefficacy of the curcumin-caffeine complex group is significantly betterthan the average of the curcumin group and the caffeine group, itindicates that curcumin and caffeine in the curcumin-caffeine complexpharmaceutical composition manifests synergy. Similarly, because thetotal dosage of the administered drug was 50 ppm, and 40% was curcuminand 60% was L-carnitine, therefore, the lipolysis efficacy of thecurcumin-L-carnitine complex group should be close to the average ofthat of the curcumin group and the caffeine group. If the lipolysisefficacy of the curcumin-L-carnitine complex group is significantlybetter than the average of the curcumin group and the L-carnitine group,it indicates that curcumin and L-carnitine in the curcumin-L-carnitinecomplex pharmaceutical composition manifests synergy.

Please refer to FIG. 13. FIG. 13 is a bar graph showing the effects ofcurcumin-other hydrophilic drug complex pharmaceutical compositions onpromoting apoptosis of mature adipocytes.

Results in FIG. 13 showed that the percentage of apoptotic cells of thesterile water control group was 0.8±0.4%, the percentage of apoptoticcells of the curcumin group was 78.4±5.4%, the percentage of apoptoticcells of the caffeine group was 2.0±1.7%, the percentage of apoptoticcells of the L-carnitine group was 1.7±0.5%, the percentage of apoptoticcells of the curcumin-caffeine complex group was 69.3±4.5%, and thepercentage of apoptotic cells of the curcumin-L-carnitine complex groupwas 74.1±10.2%.

Comparison among the apoptosis efficacy of the curcumin group, thecaffeine group, and the curcumin-caffeine complex group demonstratedthat curcumin and caffeine in the curcumin-caffeine complexpharmaceutical composition manifests synergy in lipolysis efficacy.

Comparison among the apoptosis efficacy of the curcumin group, theL-carnitine group, and the curcumin-L-carnitine complex groupdemonstrated that curcumin and L-carnitine in the curcumin-L-carnitinecomplex pharmaceutical composition manifests synergy in lipolysisefficacy.

Therefore, complex pharmaceutical compositions formed by curcumin andvarious hydrophilic drugs can all achieve the effect of lipolysis, andthere are synergies between curcumin and various hydrophilic drugs inthe effects of lipolysis. Therefore, the present invention uses curcuminand various hydrophilic drugs to prepare drug-containing micelles and asecond lipophilic drug-containing micelles, and further preparescurcumin-other hydrophilic drug complex pharmaceutical compositions,which are the pharmaceutical compositions capable of being used forlocalized lipolysis and weight reduction.

The present invention provides a preparation for a curcumin-hydrophilicdrug complex pharmaceutical composition, and the curcumin-hydrophilicdrug complex pharmaceutical composition comprises drug-containingmicelles and a hydrophilic drug; the procedure to prepare thecurcumin-hydrophilic drug complex pharmaceutical composition is asfollows:

-   -   (a5) Mixing curcumin with a solvent and stirring at 150-500 rpm        at room temperature until curcumin dissolves completely;    -   (b5) Adding a pharmaceutically acceptable surfactant and        stirring well at 100 300 rpm to volatilize the solvent, wherein,        the hydrophilic-lipophilic balance value (HLB value) of the        surfactant is greater than 10;    -   (c5) Once the solvent volatilizes completely, slowly adding a        first pharmaceutically acceptable aqueous solution and stirring        well at 100300 rpm to form drug-containing micelles and; and    -   (d5) Filtering through a 0.2 um filter, and storing the filtered        solution comprising drug-containing micelles in dark and        refrigeration;

Wherein, the first pharmaceutically acceptable aqueous solutioncomprises a hydrophilic drug.

Preferably, the first pharmaceutical composition comprises a localaesthetic.

Preferably, the local anesthetic is at least one of amides,para-aminobenzoic acid esters, and amino ethers, or combination thereof

Preferably, the amides are at least one of dibucaine, lidocaine,mepivacaine HCl, bupivacine HCl, pyrrocaine HCl, Prilocaine HCl,digammacaine, and oxethazaine, or combination thereof.

Preferably, the para-aminobenzoic acid esters are at least one ofbutacaine, dimethocaine, and tutocaine, or combination thereof.

Preferably, the amino ethers are at least one of quinisocaine andpramocaine, or combination thereof.

Preferably, the first pharmaceutically acceptable aqueous solutioncomprises an antioxidant.

Preferably, the antioxidant is at least one of beta-carotene, lutein,lycopene, bilirubin, vitamin A, vitamin C (ascorbic acid), vitamin E,uric acid, nitric oxide, nitroxide, pyruvate, catalase, superoxidedismutase, glutathione peroxidases, N-acetyl cysteine, and naringenin,or combination thereof.

Preferably, in step (a5), the boiling point of the solvent is lower thanthat of pure water.

Preferably, in step (a5), the solvent is a hydrophilic solvent.

Preferably, the hydrophilic solvent is at least one of methanol,ethanol, acetone, and other hydrophilic solvents, or combinationthereof.

Preferably, the solvent in step (a5) is a lipophilic (hydrophobic)solvent.

Preferably, the lipophilic (hydrophobic) solvent is at least one ofether, benzene, chloroform, dichloromethane, hexane, and otherlipophilic (hydrophobic) solvents, or combination thereof.

Preferably, in step (b5), the surfactant is a non-ionic surfactant.

Preferably, the non-ionic surfactant is at least one of polysorbate 80(TWEEN® 80), polyoxyl 15 hydroxystearate (KOLLIPHOR® HS 15),polyoxyethylene castor oil derivatives, and other non-ionic surfactants,or combination thereof

Preferably, the polyoxyethylene castor oil derivative is at least one ofKOLLIPHOR® ELP (polyoxyl-35-castor oil, formerly known as CREMOPHOR®ELP), KOLLIPHOR® RH 40 (polyoxyl 40 hydrogenated castor oil), and otherpolyoxyethylene castor oil derivatives, or combination thereof.

Preferably, between steps (c5) and (d5), it further includes the steps:

(c51) Adding a second pharmaceutically acceptable aqueous solution andmixing well to completely dissolve the second pharmaceuticallyacceptable aqueous solution.

Preferably, the hydrophilic drug is dissolved in the firstpharmaceutically acceptable aqueous solution, the drug-containingmicelle is a microstructure formed by the surfactant, and curcumin isencapsulated in said drug-containing micelle.

Preferably, the hydrophilic drug in the first pharmaceuticallyacceptable aqueous solution is at least one of green tea extract,epigallocatechin gallate, epicatechin, epicatechin gallate,epigallocatechin, gallocatechin gallate, gallocatechin, catechingallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine,L-carnitine, synephrine, chlorogenic acid, and other hydrophilic drugs,or combination thereof.

Preferably, in steps (a5) and (c5), the weight ratio of the curcumin andthe hydrophilic drug is 30:1 to 1:10.

Preferably, in steps (a5)-(c5), based on 1 weight unit defined as thetotal weight of the curcumin and the hydrophilic drug, the weight of thesurfactant is 0.24-70 weight units; or, the weight ratio of the totalweight of the curcumin and the hydrophilic drug to the surfactant is 4:1to 1:70.

Preferably, in steps (a5), (c5), and (c51), based on one weight unitdefined as the total weight of the curcumin and the hydrophile drug, thetotal weight of the first pharmaceutically acceptable aqueous solutionand the second pharmaceutically acceptable aqueous solution is 16-400weight units.

Preferably, in steps (c5) and (c51), the first pharmaceuticallyacceptable aqueous solution and the second pharmaceutically acceptableaqueous solution are water for injection, aqueous solution forinjection, or normal saline.

As demonstrated by the examples of the present invention, the curcuminsimple pharmaceutical composition, the curcumin-resveratrol complexpharmaceutical composition, the curcumin-green tea extract complexpharmaceutical composition, the curcumin-other lipophilic drug complexpharmaceutical composition, and the curcumin-hydrophilic drug complexpharmaceutical composition provided by the present invention, and otherpharmaceutical compositions provided by the present invention can allreduce the localized fat, and can reduce the body weight. Therefore, thecurcumin simple pharmaceutical composition, the curcumin-resveratrolcomplex pharmaceutical composition, the curcumin-green tea extractcomplex pharmaceutical composition, the curcumin-other lipophilic drugcomplex pharmaceutical composition, and the curcumin-hydrophilic drugcomplex pharmaceutical composition provided by the present invention,and other pharmaceutical compositions provided by the present inventioncan be used to prepare subcutaneous implanted devices, subcutaneousimplants, solutions for implanted infusion, cream, or patches, which iscapable of being administered at the sites requiring subcutaneous fatreduction by subcutaneous implantation, implanted infusion, cream, orpatch application. Or, the pharmaceutical compositions can be used toprepare subcutaneous implanted devices, subcutaneous implants, solutionsfor implanted infusion, cream, or patches, which is capable of beingadministered to an subject by subcutaneous implantation, intravenousinjection, implanted infusion, cream, or patch application to reduce thebody weight of the subject.

Preferably, the curcumin simple pharmaceutical composition, thecurcumin-resveratrol complex pharmaceutical composition, thecurcumin-green tea extract complex pharmaceutical composition, thecurcumin-other lipophilic drug complex pharmaceutical composition, andthe curcumin-hydrophilic drug complex pharmaceutical compositionprovided by the present invention, and other pharmaceutical compositionsprovided by the present invention can reduce the fat at theadministration site or the body weight by subcutaneous fat injection.Thus, the curcumin simple pharmaceutical composition, thecurcumin-resveratrol complex pharmaceutical composition, thecurcumin-green tea extract complex pharmaceutical composition, thecurcumin-other lipophilic drug complex pharmaceutical composition, andthe curcumin-hydrophilic drug complex pharmaceutical composition, andother pharmaceutical compositions provided by the present invention canbe used to prepare subcutaneous fat layer injection formulation orsubcutaneous injection formulation for reducing localized subcutaneousfat.

Preferably, the curcumin simple pharmaceutical composition, thecurcumin-resveratrol complex pharmaceutical composition, thecurcumin-green tea extract complex pharmaceutical composition, thecurcumin-other lipophilic drug complex pharmaceutical composition, andthe curcumin-hydrophilic drug complex pharmaceutical compositionprovided by the present invention, and other pharmaceutical compositionsprovided by the present invention can reduce the body weight bysubcutaneous fat injection formulation or intravenous injection. Thus,the curcumin simple pharmaceutical composition, the curcumin-resveratrolcomplex pharmaceutical composition, the curcumin-green tea extractcomplex pharmaceutical composition, the curcumin-other lipophilic drugcomplex pharmaceutical composition, and the curcumin-hydrophilic drugcomplex pharmaceutical composition provided by the present invention,and other pharmaceutical compositions provided by the present inventioncan be used to prepare subcutaneous fat layer injection formulation,intravenous injection formulation, and subcutaneous injectionformulation for reducing body weight.

The foregoing descriptions are merely the preferred embodiments of thepresent invention and are not intended to limit the scope of patentapplication of the present invention. Therefore, any alteration ormodification that does not depart from the spirits disclosed hereinshould be included within the scope of patent application of the presentinvention.

1-22. (canceled)
 23. A method for reducing body weight of a subject,comprising administering a pharmaceutical composition to the subject,wherein the pharmaceutical composition comprises: a first plurality ofdrug-containing micelles; and a curcuminoid, which is encapsulated insaid first plurality of drug-containing micelles; wherein the firstplurality of drug-containing micelles has a microstructure formed by afirst pharmaceutically acceptable non-ionic surfactant, which has ahydrophilic-lipophilic balance (HLB) value greater than
 10. 24. Themethod of claim 23, wherein the pharmaceutical composition furthercomprises a pharmaceutically acceptable aqueous solution.
 25. The methodof claim 23, wherein the pharmaceutical composition further comprises asecond plurality of lipophilic drug-containing micelles; wherein thesecond plurality of lipophilic drug-containing micelles has a secondmicrostructure formed by a second non-ionic surfactant, and a lipophilicdrug, which is encapsulated in said second plurality of lipophilicdrug-containing micelles, and the lipophilic drug comprises quercetin,synephrine, puerarin, resveratrol, or a combination thereof.
 26. Themethod of claim 23, wherein the pharmaceutical composition furthercomprises a hydrophilic drug, which comprises green tea extract,epicatechin, epicatechin gallate, epigallocatechin, gallocatechingallate, gallocatechin, catechin gallate, catechin, epigallocatechingallate (EGCG), caffeine, carnitine, L-carnitine, synephrine,chlorogenic acid, or a combination thereof.
 27. The method of claim 23,wherein the pharmaceutical composition is administered to the subject bysubcutaneous injection, intravenous injection, or subcutaneous fat layerinjection.
 28. The method of claim 27, wherein the pharmaceuticalcomposition is injected at a local site of the subject at a dose of0.2˜16 mg/cm².
 29. The method of claim 27, wherein the pharmaceuticalcomposition is injected at a local site of the subject at a dose of0.4˜40 mg/kg.
 30. The method of claim 27, wherein the pharmaceuticalcomposition is administered at a local site of the subject at afrequency of 3-60 times every other day to every 20 days.
 31. The methodof claim 23, wherein the pharmaceutical composition further comprises acosolvent, a suspending agent, an oil phase excipient, or a combinationthereof.
 32. The method of claim 31, wherein the pharmaceuticalcomposition further comprises the cosolvent and the oil phase excipient;and wherein the first plurality of drug-containing micelles is co-formedwith the first non-ionic surfactant and at least one of the oil phaseexcipient and the cosolvent.
 33. The method of claim 23, wherein thepharmaceutical composition is a subcutaneous injection formulation, asubcutaneous fat layer injection formulation, an intravenous injectionformulation, a solution for implanted infusion, a cream, or a patch. 34.The method of claim 23, wherein the first non-ionic surfactant comprisespolysorbate 80, polyxyl 15 hydroxystearate, a polyoxyethylene castor oilderivative, or a combination thereof.
 35. The method of claim 34,wherein the first non-ionic surfactant comprises one or morepolyoxyethylene castor oil derivatives.
 36. The method of claim 35,wherein the polyoxyethylene castor oil derivative comprises polyoxyl 35castor oil, polyoxyl 40 hydrogenated castor oil, or a combinationthereof.
 37. The method of claim 23, wherein the weight ratio of thecurcuminoid to the first non-ionic surfactant in the first plurality ofdrug-containing micelles is 1:8 to 1:500.
 38. The method of claim 23,wherein the curcuminoid is curcumin.
 39. The method of claim 23, whereinthe diameter of the drug-containing micelles is 3-50 nm.
 40. The methodof claim 25, wherein the second non-ionic surfactant has an HLB valuegreater than
 10. 41. The method of claim 40, wherein the secondnon-ionic surfactant comprises polysorbate 80, polyoxyl 15hydroxystearate, polyoxyethylene castor oil derivatives, or acombination thereof.
 42. The method of claim 41, wherein the secondnon-ionic surfactant comprises polyoxyl 35 castor oil, polyoxyl 40hydrogenated castor oil, or a combination thereof.
 43. The method ofclaim 23, wherein the concentration of the curcuminoid in thepharmaceutical composition is 0.3˜120 mg/g.
 44. The method of claim 25,wherein the weight ratio of the curcuminoid to the lipophilic drug is30:1˜1:10.
 45. The method of claim 26, wherein the weight ratio of thecurcuminoid to the hydrophilic drug is 30:1 to 1:10.